"Aseptic Technique". In: Current Protocols in Microbiology
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Aseptic Technique APPENDIX 4D Tomasz Bykowski 1 and Brian Stevenson2 1Center for Medical Education, Warsaw, Poland 2Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky ABSTRACT This chapter describes common laboratory procedures that can reduce the risk of culture con- taminations (sepsis), collectively referred as “aseptic technique.” Two major strategies of aseptic work are described: using a Bunsen burner and a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microor- ganisms on petri dishes. Curr. Protoc. Microbiol. 11:A.4D.1-A.4D.11. C 2008 by John Wiley & Sons, Inc. Keywords: aseptic technique r sterilization r bacteriology r Bunsen burner r laminar flow r laminar hood r virology r cell culture INTRODUCTION understand the appropriate methods of dis- Aseptic technique is a set of routine mea- posal and decontamination for the specific mi- sures that are taken to prevent cultures, sterile croorganism with which he or she is working. media stocks, and other solutions from being Refer to UNIT 1A.1 and APPENDIX 1B for more contaminated by unwanted microorganisms information. (i.e., sepsis). While such actions are some- times called “sterile technique,” that terminol- The Bunsen Burner ogy is appropriate only in reference to prevent- Probably the easiest way to create a ing introduction of any organisms to labora- relatively sterile environment on the lab- tory or medical equipment and reagents (e.g., oratory bench is by using a simple gas- during surgery). Since the goal of a biologist powered burner (see Fig. A.4D.1). This com- is to grow microorganisms or eukaryotic cells mon piece of equipment burns a continuous without introduction of extraneous organisms, stream of a flammable gas—usually natural aseptic techniques are crucial for accurate and gas (methane)—based upon a design made al- meaningful experimentation. most 150 years ago by the German chemist One should always remember that a com- Robert Wilhelm Bunsen (1811-1899). A ma- pletely sterile working environment does not jor purpose of the open flame in aseptic tech- exist. However, there are a number of sim- nique is to create a cone of hot air above and ple, common sense procedures that will reduce around the laboratory bench to reduce the vi- the risk of culture contaminations. Examples ability of organisms on suspended dust parti- of aseptic technique are cleaning and disin- cles. The ability of the Bunsen burner flame to fecting lab surfaces prior to use, limiting the heat things very quickly also makes it an ideal duration that cultures or media are uncapped choice for sterilizing inoculating loops, warm- and exposed to the air, keeping petri dishes ing glass bottle necks, or igniting alcohol on closed whenever possible, effectively steriliz- culture spreaders. ing inoculating loops and other equipment that A Bunsen burner is not practical in some sit- comes into contact with cultures or media, and uations, e.g., within a laminar flow unit where avoiding breathing on cultures or sterile instru- the heat will disrupt airflow. A microincinera- ments. These precautions will become second tor may be used as an alternative. This con- nature after practical laboratory experience. sists of a circular heating element. Placing CAUTION: The information in this an inoculating loop or needle within the ring APPENDIX is meant as an introduction to basic will quickly heat and sterilize the loop/needle. aseptic technique and does not cover safety Note that a microincinerator does not provide aspects necessary for working with specific other aseptic technique benefits of a lit Bunsen Commonly Used Methods for Cell organisms. It is important that the reader burner. Culture Current Protocols in Microbiology A.4D.1-A.4D.11, November 2008 A.4D.1 Published online November 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/9780471729259.mca04ds11 Supplement 11 Copyright C 2008 John Wiley & Sons, Inc. chimney air regulating collar gas rubber tubing Figure A.4D.1 Bunsen burner. The gas burner consists of a vertical metal tube through which a narrow jet of natural gas is directed. Air is drawn in via air holes located near the stand. The gas/air mixture burns above the upper opening. The regulating collar can be turned to cover or partly cover the air holes, which allows regulation of the amount of air sucked in and hence the temperature and shape of the flame. The Laminar Flow Unit laboratories, where they help prevent spread A laminar flow unit (or hood) is a sophis- of viruses and some bacteria. Consult UNIT 1A.1 ticated appliance that can further help pre- as well as the resources in APPENDIX 1B vent contamination of reagents and biological for additional information on biosafety and cultures. Used correctly, it provides the work biocontainment. space with clean, ultrafiltered air. It also keeps room air from entering the work area and both suspends and removes airborne contaminants STRATEGIC PLANNING introduced into the work area by personnel. Contamination Risk Assessment and The most important part of a laminar flow Space Organization hood is a high-efficiency bacteria-retentive fil- Localizing potential sources of biological ter, i.e., the HEPA (high-efficiency particulate contamination is an important part of arrang- air) filter. A certified HEPA filter must cap- ing space when a room is used as a laboratory. ture a minimum of 99.97% of dust, pollen, Benches for aseptic work should be organized mold, bacteria, and any airborne particles with away from air conditioning vents, cooling asizeof>0.3 μm at 85 liters/min. The first fans, open windows, or blowers from heating HEPA filters were developed in the 1940s by or refrigerating systems. Containers used for the U.S.A. Atomic Energy Commission as part the disposal of biological material may con- of the Manhattan Project (the development of tain large numbers of viable microorganisms, the atomic bomb) to provide an efficient, effec- even when emptied. Some contaminants, e.g., tive way to filter radioactive particulate con- sporulating bacteria, yeasts, or fungi, are very taminants. HEPA filter technology was declas- difficult to eradicate from the working envi- sified after World War II, allowing extensive ronment. Waste should be kept very well cov- research and commercial use. ered or, if feasible, in a different room, espe- Laminar flow hoods are essential com- cially if using particularly harmful or sturdy ponents of many biosafety level (BSL)-2 microorganisms. Aseptic Technique A.4D.2 Supplement 11 Current Protocols in Microbiology General Cleanliness that are either flammable or would be de- In principle, keeping high standards of natured by heat are usually filter sterilized. cleanliness has to be a universally accepted A range of syringe-based or bottle-top fil- norm in the biological laboratories. People ters of different pore sizes physically remove should wear lab coats that are worn only in (exclude) living organisms and can be used the laboratory. Researchers should wash their according to the suppliers’ instructions. Filtra- hands often during the course of the day, es- tion devices may use either negative (vacuum) pecially prior to any and all handling of bi- or positive pressure. An advantage of positive ological cultures, media, or sterile supplies. pressure in filtering is that it reduces clogging Dust and stains must be removed regularly, from precipitated salts. and spills cleaned and decontaminated im- Most living organisms are retained by a mediately. Bench tops and shelves should be 0.45-μm filter. That size filter is often used washed immediately before all uses with 10% as a prefilter because it also clears the liquid (v/v) household bleach (containing sodium of larger particulates. However, because many hypochlorite). This solution will inactivate all bacteria can pass through 0.45-μm pores, a viruses, bacteria, fungi, and other potential 0.22-μm filter should be used to ensure steril- contaminants. Alternatively, a solution of an- ization of the fluids. Viruses will pass through tiseptic cleanser (e.g., Lysol), diluted accord- 0.22-μm filters, although they generally stick ing to the manufacturer’s directions, is ade- to the membrane. Some bacteria, such as quate. Organic disinfectants such as 70% (v/v) saprophytic Leptospira spp. commonly found ethanol are generally less effective than bleach in domestic water supplies, may also pass solutions because ethanol may evaporate too through 0.22 μm filters. If such potential quickly to effectively sterilize surfaces and contaminants may cause difficulties, consider may not completely inactivate all potential autoclaving water before using for solutions contaminants. Outerwear may be covered with that need to be filter sterilized. dirt or dust, and so should be kept in a cloak- Filtering procedures are usually carried out room away from the work space. These simple in a laminar flow unit or on a bench equipped rules of cleanliness will eliminate the bulk of with Bunsen burner (see below) to avoid potential contaminations from the work area. recontamination of just-sterilized material. Sterilizing Equipment and Reagents as Autoclaving a Prelude to Effective Aseptic Work CAUTION: Items containing volatile sol- Starting aseptic work with material that vents or corrosive chemicals (e.g., phenol, is contaminated is a path to nowhere. Many trichloroacetic acid, ether, chloroform), or reagents are supplied by their manufacturer as any radioactive isotopes cannot be autoclaved ready to use for cultures, while many others under any circumstances. do not have to be sterilized at all (e.g., ethanol, Autoclaving (using steam under pressure) phenol, or concentrated detergents). How- is a rapid method for sterilizing almost any- ever, some require sterilization (i.e., the com- thing except heat-labile substances.