《英语教学》（英文版）Working Safely with Biological Samples.pdf

What Are the Necessary Precautions and Differences in Handling of Viruses, Bacteria, Fungi, and Protozoa? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 What Precautions Should Be Taken with Experimental Animals? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 What Precautions Should Be Considered before and during the Handling of Human Tissues and Body Fluids? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 What Is the Best Way to Decontaminate Your Work Area after Taking down Your Experiment? . . . . . . . . . . . . 130 Is It Necessary to Decontaminate Yourself or Your Clothing? Is There Significant Risk of Contaminating Others? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Media Preparation and Sterilization . . . . . . . . . . . . . . . . . . . . . . 132 How Can You Work Most Efficiently with Your Media Preparation Group? . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Which Autoclave Settings Are Appropriate for Your Situation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 What Is the Best Wrapping for Autoclaving? Aluminum Foil, Paper, or Cloth? . . . . . . . . . . . . . . . . . . . . 134 What Are the Time Requirements of Autoclaving? . . . . . . . 135 What If the Appearance of the Indicator Tape Didn’t Change during Autoclaving? . . . . . . . . . . . . . . . . . . . 135 Why Is Plastic Labware Still Wet after Applying the Dry Cycle? Is Wet Labware Sterile? . . . . . . . . . . . . . . . . . 135 Can Your Plastic Material Be Sterilized? . . . . . . . . . . . . . . . . . 135 Requesting the Media Room to Sterilize Labware . . . . . . . . 136 Requesting the Media Room to Prepare Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 Allow Sufficient Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Autoclaving for the Do-It-Yourselfer . . . . . . . . . . . . . . . . . . . 137 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 BIOSAFETY Is There Such a Thing as a Nonpathogenic Organism? The term “biohazard” is applied to any living agent that has the potential to cause infection and disease if introduced into a suitable host in an infectious dose. The “living agent” can include viruses, bacteria, fungi, protozoa, helminths (worms) and their eggs or larvae, and arthropods (insects, crustaceans) and their eggs or larvae. We most commonly think of pathogenic microbes such as Salmonella typhi and Leishmania donovani as biohazards, but if introduced into a healthy body in large numbers (or an immuno- 114 Haidaris and Brownlow

compromised body in low numbers), organisms that are normally nonpathogenic can cause infection. The infectious dose will vary with the organism and the health of those infected. For example, Shigella flexneri ree the ingestion of only a few hundred organisms to cause intestinal disease. Salmonella typhi requires over a hundredfold more organisms to do so. Technically there is no such thing as a nonpathogenic microorganism Do You know the biohazard safety Level of Your research materials? Regardless of the type of work you will be doing with micro- organisms, it is mandatory to know as much as possible about the safety precautions needed to handle the microbes you will be using, prior to entering the lab. Does your organism requir special handling? Ask questions of the lab supervisor and your co- workers regarding the microbe itself, its safe handling and proper disposal. Know the location of first-aid kits, eyewash stations, and biosafety level(BSl) required to contain them, with BSL-1 being the lowest and BSL-4 being the highest levels, respectively. These classifications have been set by the U.S. government agencies such as the Centers for Disease Control and Prevention(CDC)in Atlanta, Georgia, and its associated institution, the National Center for Infectious Diseases(NCID), the World Health Organi zation(WHO), and the governments of the European Community (EC). The CDC and NCID are an excellent source of information on the biosafety level classification of individual organisms, and the methods suggested for their safe handling. The CDC Web site is at www.cdcgov,whichhaslinkstotheNcidsiteanOtherexcellent source of information is a book entitled laboratory acquired Infections(C A Collins and D A Kennedy, 1999) BSL- and BSL-2 BSL-1 agents present no, or minimal, hazard under ordinary conditions of safe handling. The common host cells used in cloning experiments are classified as BSL-1, such as E coli and Saccha romyces cerevisiae, and they can be handled at the benchto Simple disinfection of the workbench after handling and good hand-washing will be sufficient to eliminate organisms from any spillage. Low-level pathogens such as the fungus Candida albicans or the bacterium Staphylococcus aureus can also be safely handled at the benchtop, if the organism does not come into contact with Working Safely with Biological Samples

compromised body in low numbers), organisms that are normally nonpathogenic can cause infection. The infectious dose will vary with the organism and the health of those infected. For example, Shigella flexneri requires the ingestion of only a few hundred organisms to cause intestinal disease. Salmonella typhi requires over a hundredfold more organisms to do so. Technically there is no such thing as a nonpathogenic microorganism. Do You Know the Biohazard Safety Level of Your Research Materials? Regardless of the type of work you will be doing with microorganisms, it is mandatory to know as much as possible about the safety precautions needed to handle the microbes you will be using, prior to entering the lab. Does your organism require special handling? Ask questions of the lab supervisor and your coworkers regarding the microbe itself, its safe handling and proper disposal. Know the location of first-aid kits, eyewash stations, and emergency lab showers. In general, organisms used in the lab are classified in terms of the biosafety level (BSL) required to contain them, with BSL-1 being the lowest and BSL-4 being the highest levels, respectively. These classifications have been set by the U.S. government agencies such as the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, and its associated institution, the National Center for Infectious Diseases (NCID), the World Health Organization (WHO), and the governments of the European Community (EC). The CDC and NCID are an excellent source of information on the biosafety level classification of individual organisms,and the methods suggested for their safe handling.The CDC Web site is at www.cdc.gov, which has links to the NCID site. Another excellent source of information is a book entitled Laboratory Acquired Infections (C. A. Collins and D. A. Kennedy, 1999). BSL-1 and BSL-2 BSL-1 agents present no, or minimal, hazard under ordinary conditions of safe handling.The common host cells used in cloning experiments are classified as BSL-1, such as E. coli and Saccharomyces cerevisiae, and they can be handled at the benchtop. Simple disinfection of the workbench after handling and good hand-washing will be sufficient to eliminate organisms from any spillage. Low-level pathogens such as the fungus Candida albicans or the bacterium Staphylococcus aureus can also be safely handled at the benchtop, if the organism does not come into contact with Working Safely with Biological Samples 115

he skin. or mucous membranes. Care should also be taken when handling sharps that may be contaminated with a microbe A contaminated scalpel blade, needle, or broken glass can serve as a vehicle of entry to the body. As described more thoroughly wearing a lab coat, safety glasses, and disposable gloves is always a good idea when handling any quantity of microorgan- ism. The handling of liquid culture could also lead to aerosoliza- tion of the microbial suspension, hence wearing a particle mask might be useful as a precaution. It is always best to take an approach that maximizes your own safety and the safety of those BSL-2 agents possess the potential for biohazard, and they may oduce dis de of severity as a result of accidental laboratory infections. Moderate level pathogens, such as Neisseria gonorrhoeae, are classified as BSL-2. A safe way to handle BsL-2 organisms is in a laminar airflow, biosafety hood By generating a flow of air inside the cabinet, these hoods are designed to keep aerosols from leaving the hood and entering the rooms airspace. The hoods proper function should be certi fied yearly by professionals. The Environmental Health and Safety officers of your institution or outside contractors can perform this function Usually a germicidal UV light is used to disinfect the inside of the hood when not in use. As a precaution prior to use of th hood, the airflow should be on for 15 minutes while the germici dal light is on A wipe-down of the hoods inside working surface with 70% ethanol is also a useful precaution after the UV light is turned off. The airflow should be kept on during the entire time the hood is in use, and the glass panel on the front of the hood be raised only high enough fortable of the worker's arms inside the hood. Most hoods are equipped with an alarm to warn the worker if the front panel is raised too high Basic microbiological techniques of sterility, a minimum of protective gear, disinfectant, and common sense are all that are required to safely handle BsL-l microbes used as cloning or expression vectors in the laboratory. Under common sense and in accordance with safety regulations, there should be absolutely no eating or drinking by an individual during the handling of a microorganism in the lab. Those workers in a diagnostic microbiology lab or doing research on a BSL-2 pathogen will wish to use a biosafety hood when necessary, along with protective clothing I 16 Haidaris and Brownlow

the skin, or mucous membranes. Care should also be taken when handling sharps that may be contaminated with a microbe. A contaminated scalpel blade, needle, or broken glass can serve as a vehicle of entry to the body. As described more thoroughly below, wearing a lab coat, safety glasses, and disposable gloves is always a good idea when handling any quantity of microorganism. The handling of liquid culture could also lead to aerosolization of the microbial suspension, hence wearing a particle mask might be useful as a precaution. It is always best to take an approach that maximizes your own safety and the safety of those around you. BSL-2 agents possess the potential for biohazard, and they may produce disease of varying degrees of severity as a result of accidental laboratory infections. Moderate level pathogens, such as Neisseria gonorrhoeae, are classified as BSL-2. A safe way to handle BSL-2 organisms is in a laminar airflow, biosafety hood. By generating a flow of air inside the cabinet, these hoods are designed to keep aerosols from leaving the hood and entering into the room’s airspace. The hood’s proper function should be certi- fied yearly by professionals.The Environmental Health and Safety officers of your institution or outside contractors can perform this function. Usually a germicidal UV light is used to disinfect the inside of the hood when not in use. As a precaution prior to use of the hood, the airflow should be on for 15 minutes while the germicidal light is on. A wipe-down of the hood’s inside working surface with 70% ethanol is also a useful precaution after the UV light is turned off. The airflow should be kept on during the entire time the hood is in use, and the glass panel on the front of the hood should be raised only high enough to allow comfortable use of the worker’s arms inside the hood. Most hoods are equipped with an alarm to warn the worker if the front panel is raised too high. Basic microbiological techniques of sterility, a minimum of protective gear, disinfectant, and common sense are all that are required to safely handle BSL-1 microbes used as cloning or expression vectors in the laboratory. Under common sense and in accordance with safety regulations, there should be absolutely no eating or drinking by an individual during the handling of a microorganism in the lab. Those workers in a diagnostic microbiology lab or doing research on a BSL-2 pathogen will wish to use a biosafety hood when necessary, along with protective clothing. 116 Haidaris and Brownlow

bSL-3 and bSL-4 Any organism requiring BSL-3 or BSL-4 containment should only be handled by highly trained individuals, using extensiv safety precautions. Training in a lab that has experience with the microbe is highly recommended. BSL-3 pathogens pose special hazards to laboratory workers They must be handled, at very least, in a biosafety hood. No open containers or those with the potential to break easily should leave the hood if they contain the bsl-3 agent. Eye protection, particle mask, lab coat, and gloves are mandatory. Centrifugation of such organisms requires sealed containers to prevent aerosol and Virulent BSL-3 pathogens that cause disease in low numbers and are transmitted by aerosol, such as Mycobacterium tuber culosis, require environmentally sealed containment rooms and also require the worker to be completely protected by special clothing, colloquially referred to as a"moonsuit, because it resembles the type worn by astronauts. Disinfection and re moval of the suit is required before the wearer can enter the open environment BSL-4 pathogens pose an extremely serious hazard to the lab oratory worker. These are the"hottest"pathogens, such as Ebola virus. Only a few places in the United States and elsewhere in the world are equipped for such studies. Only highly trained profes- sionals are qualified to handle these agents How Can You Learn More about the Genealogy of Your host cells? If the cell is obtained from a commercial source. such as a biotechnology company or the American Type Culture Collection then the background on the host cell is often provided with the cell stock or in the catalog of the company For strains of E. coli commonly used for cloning, the catalogs of biotech nology companies often have appendixes that list phenotypes for the given strains. If the cell from a personal contact (i.e, another scientist), then be sure to ask for references or technical material on the cultivation and use of the cell. If possible, try to reproduce the desired cellular activity in a small pilot experiment prior to using precious materials or resources in a large-scale study. A wealth of genetic information and links to e. coli resources is available at httpcgscbiologyyaleedul. Working Safely with Biological Samples 117

BSL-3 and BSL-4 Any organism requiring BSL-3 or BSL-4 containment should only be handled by highly trained individuals, using extensive safety precautions. Training in a lab that has experience with the microbe is highly recommended. BSL-3 pathogens pose special hazards to laboratory workers. They must be handled, at very least, in a biosafety hood. No open containers or those with the potential to break easily should leave the hood if they contain the BSL-3 agent. Eye protection, particle mask, lab coat, and gloves are mandatory. Centrifugation of such organisms requires sealed containers to prevent aerosol and spillage. Virulent BSL-3 pathogens that cause disease in low numbers and are transmitted by aerosol, such as Mycobacterium tuberculosis, require environmentally sealed containment rooms and also require the worker to be completely protected by special clothing, colloquially referred to as a “moonsuit,” because it resembles the type worn by astronauts. Disinfection and removal of the suit is required before the wearer can enter the open environment. BSL-4 pathogens pose an extremely serious hazard to the laboratory worker. These are the “hottest” pathogens, such as Ebola virus. Only a few places in the United States and elsewhere in the world are equipped for such studies. Only highly trained professionals are qualified to handle these agents. How Can You Learn More about the Genealogy of Your Host Cells? If the cell is obtained from a commercial source, such as a biotechnology company or the American Type Culture Collection, then the background on the host cell is often provided with the cell stock or in the catalog of the company. For strains of E. coli commonly used for cloning, the catalogs of biotechnology companies often have appendixes that list phenotypes and original references for the given strains. If the cell comes from a personal contact (i.e., another scientist), then be sure to ask for references or technical material on the cultivation and use of the cell. If possible, try to reproduce the desired cellular activity in a small pilot experiment prior to using precious materials or resources in a large-scale study. A wealth of genetic information and links to E. coli resources is available at http://cgsc.biology.yale.edu/. Working Safely with Biological Samples 117