Are You Properly Dressed and Equipped for Lab work? Regardless of the level of one's experience in the laboratory, it is wise to be prepared for the worst in terms of accidents. Proper preparation starts with proper protection for yourself and your co-workers. a number of common accessories should be used as needed Lab coat Most laboratories require the wearing of a lab coat, but even when not mandatory, a lab coat is a good idea. Protection of cloth ing and, more important, the skin underneath, is worth the effort and (perceived)inconvenience For those working with microbes, clothing can be permanently fouled by a spill of almost any microorganism. Even a small break in the skin can serve as a portal of entry for a seemingly innocuous microbe that can result in a serious infection if the microbes gain access to the circulation in sufficient numbers. For those working with flames, such as Bunsen burner, some institutions require a lab coat of flame retar- dant material. This ensures an added level of protection should there be a spill of a flammable liquid followed by ignition at the lab bench Closed footwear n The open-toed shoe, sandal, or"flip-flop, even while wearing s, provides easy access to the foot for sharps, hazardous chemicals, and infectious agents. The protection afforded by closed-toe shoe against these assaults could provide an important level of safety. When choosing between fashion, or even comfort and protection, protect your feet! Eye Protection 4. It is strongly suggested to wear some form of eye protection when handling large volumes of microorganisms to protect against splashing during handling. Even a nonpathogen can set up infec tion when introduced in large numbers on the conjunctiv Eyeglass(but not contact lens) wearers are afforded reasonable protection in these circumstances, but more safety is provided with the larger protective surface afforded by safety goggles or glasses, which can fit over conventional eyeglasses if necessary. For indi viduals working with pathogenic organisms, it is essential to wear eye protection at all times Conventional safety glasses are suitable against BSL-1 BSL-2 class pathogens For BSL-3 pathogens, a full-length Haidaris and Brownlow
Are You Properly Dressed and Equipped for Lab Work? Regardless of the level of one’s experience in the laboratory, it is wise to be prepared for the worst in terms of accidents. Proper preparation starts with proper protection for yourself and your co-workers. A number of common accessories should be used as needed. Lab Coat Most laboratories require the wearing of a lab coat, but even when not mandatory, a lab coat is a good idea. Protection of clothing and, more important, the skin underneath, is worth the effort and (perceived) inconvenience. For those working with microbes, clothing can be permanently fouled by a spill of almost any microorganism. Even a small break in the skin can serve as a portal of entry for a seemingly innocuous microbe that can result in a serious infection if the microbes gain access to the circulation in sufficient numbers. For those working with flames, such as a Bunsen burner, some institutions require a lab coat of flame retardant material. This ensures an added level of protection should there be a spill of a flammable liquid followed by ignition at the lab bench. Closed Footwear The open-toed shoe, sandal, or “flip-flop,” even while wearing socks, provides easy access to the foot for sharps, hazardous chemicals, and infectious agents. The protection afforded by a closed-toe shoe against these assaults could provide an important level of safety. When choosing between fashion, or even comfort, and protection, protect your feet! Eye Protection It is strongly suggested to wear some form of eye protection when handling large volumes of microorganisms to protect against splashing during handling. Even a nonpathogen can set up infection when introduced in large numbers on the conjunctiva. Eyeglass (but not contact lens) wearers are afforded reasonable protection in these circumstances, but more safety is provided with the larger protective surface afforded by safety goggles or glasses, which can fit over conventional eyeglasses if necessary. For individuals working with pathogenic organisms, it is essential to wear eye protection at all times. Conventional safety glasses are suitable against BSL-1 and BSL-2 class pathogens. For BSL-3 pathogens, a full-length pro- 118 Haidaris and Brownlow
tective face mask will provide additional defense against acciden tal exposure of the face and eyes Latex gloves The wearing of protective gloves is a good idea if the skin on the hands is abraded or raw. It is very important to wear protec- tive gloves if a pathogen is being handled. Some people's hands can be irritated by the powder on many types of latex gloves. Most can be obtained in powder-free form. Less fortunate individuals are allergic to the latex in the gloves. Cloth glove inserts are available that can prevent contact of the latex with skin Heavy-Duty Protective Glove For those individuals handling pathogens and sharps simulta neously, such as during inoculation of an experimental animal, it is a good idea to wear a heavier rubber glove over the latex glove. Removal of the last 0.5 to 1 inch of the finger tips of the heavy glove will afford the worker with the dexterity to handle instru ments or other items with efficiency, and still provide protection to the bulk of the hand Safety e nt and Supplies The two most important pieces of safety equipment in any lab are the eyewash station and the emergency lab shower. These two equipment stations months)to be sure they are fully operative Know their location In the event of the splashing of a microbial suspension in the eye, if possible, go directly to the eyewash station and flush the eye thoroughly with water. Then seek medical attention immediately Even small numbers of microbes can permanently damage the eye if it is left untreated. The lab shower is very useful in the case of a chemical spill, and a large spill of a serious pathogen on the body shower.Again, seek medical attention folon sing under the lab stance. Finally, every lab should have a well-stocked first-aid kit for treatment of minor mishaps, and to provide intermediate care for more serious accidents. Take it upon yourself, or appoint a lab safety officer, to be sure the first-aid kit is stocked and the wash stations are operative Are You Aware of the Potential Hazards during the setup, Execution, and Cleanup of the Planned Experiment? In the microbiology lab there are physical, chemical, and micro- bial hazards. When handling a hazardous material or performing Working Safely with Biological Samples
tective face mask will provide additional defense against accidental exposure of the face and eyes. Latex Gloves The wearing of protective gloves is a good idea if the skin on the hands is abraded or raw. It is very important to wear protective gloves if a pathogen is being handled. Some people’s hands can be irritated by the powder on many types of latex gloves. Most can be obtained in powder-free form. Less fortunate individuals are allergic to the latex in the gloves. Cloth glove inserts are available that can prevent contact of the latex with skin. Heavy-Duty Protective Gloves For those individuals handling pathogens and sharps simultaneously, such as during inoculation of an experimental animal, it is a good idea to wear a heavier rubber glove over the latex glove. Removal of the last 0.5 to 1 inch of the finger tips of the heavy glove will afford the worker with the dexterity to handle instruments or other items with efficiency, and still provide protection to the bulk of the hand. Safety Equipment and Supplies The two most important pieces of safety equipment in any lab are the eyewash station and the emergency lab shower. These two equipment stations should be regularly tested (every 6–12 months) to be sure they are fully operative. Know their location. In the event of the splashing of a microbial suspension in the eye, if possible, go directly to the eyewash station and flush the eye thoroughly with water. Then seek medical attention immediately. Even small numbers of microbes can permanently damage the eye if it is left untreated. The lab shower is very useful in the case of a chemical spill, and a large spill of a serious pathogen on the body surface also could be removed in part by rinsing under the lab shower. Again, seek medical attention following such a circumstance. Finally, every lab should have a well-stocked first-aid kit for treatment of minor mishaps, and to provide intermediate care for more serious accidents. Take it upon yourself, or appoint a lab safety officer, to be sure the first-aid kit is stocked and the wash stations are operative. Are You Aware of the Potential Hazards during the Setup, Execution, and Cleanup of the Planned Experiment? In the microbiology lab there are physical, chemical, and microbial hazards. When handling a hazardous material or performing Working Safely with Biological Samples 119
a hazardous procedure, the most important thing is to pay atten- tion to what you are doing and, if transporting the material, where you are going. Wear the protective clothing as outlined in the previous section. Do not stop to answer the phone or chat. No clowning around; this is not a time for levity. Even an act as simple as sterilizing an inoculating loop can be hazardous if you get The physical hazards include burns and cuts from sharps. Burns can result from a Bunsen burner or gas jet, an inoculating loop, a hot plate, or the autoclave. When handling items going in or coming out of the autoclave, heavy-duty cloth gloves designed for handling hot containers are essential Materials heated on a hot plate or in a boiling water bath should also be handled with heav duty protective gloves. Burns can also result from ignition of flammables like ethanol or acetone. Always keep containers with these liquids safely away from a heat source. Malfunctioning machinery can also be a source of a burn or a fire. a pump motor that has seized can cause a fire. If you smell smoke or other toxic gases emanating from a piece of equipment try to turn it off or unplug it immediately, and call the fire department if necessary. If this action seems unsafe, call the fire department immediatelⅤ Needles, broken glass, scalpels, and razor blades are all poten tial hazards. Pay attention when handling them and dispose of hem properly. Most all labs require disposal using a certified arps"container, and removal by housekeeping staff or health safety workers. Never throw a sharp into the everyday trash This is a potential hazard and possible source of infection for the housekeepers. Nearly all microbiology labs utilize corrosive acids, alkalis, and organic compounds that are toxic. The potential for toxicity can manifest itself through amounts as small as the fumes released by opening the container. Even a whiff of a concentrated acid or other corrosive liquid can cause tissue damage to the naso- pharynx. A spill of even a few hundred milliliters of an organic chemical, like phenol the body can be life-threate Phenol vapors, in excessive amounts, can cause damage to the nasopharyngeal mucosa and to the mucous membranes of the eye. Brief exposure to phenol vapors can cause minor irritation of these mucosa as well. If one uses phenol frequently, it is sensible to perform the manipulations in a chemical fume hood if possible. Sources of microbial contamination to you and others are aerosols formed by the handling of the inoculating loop, prepara Haidaris and Brownlow
a hazardous procedure, the most important thing is to pay attention to what you are doing and, if transporting the material, where you are going. Wear the protective clothing as outlined in the previous section. Do not stop to answer the phone or chat. No clowning around; this is not a time for levity. Even an act as simple as sterilizing an inoculating loop can be hazardous if you get distracted. The physical hazards include burns and cuts from sharps. Burns can result from a Bunsen burner or gas jet, an inoculating loop, a hot plate, or the autoclave. When handling items going in or coming out of the autoclave, heavy-duty cloth gloves designed for handling hot containers are essential. Materials heated on a hot plate or in a boiling water bath should also be handled with heavy duty protective gloves. Burns can also result from ignition of flammables like ethanol or acetone. Always keep containers with these liquids safely away from a heat source. Malfunctioning machinery can also be a source of a burn or a fire. A pump motor that has seized can cause a fire. If you smell smoke or other toxic gases emanating from a piece of equipment try to turn it off or unplug it immediately, and call the fire department if necessary. If this action seems unsafe, call the fire department immediately. Needles, broken glass, scalpels, and razor blades are all potential hazards. Pay attention when handling them and dispose of them properly. Most all labs require disposal using a certified “sharps” container, and removal by housekeeping staff or health safety workers. Never throw a sharp into the everyday trash. This is a potential hazard and possible source of infection for the housekeepers. Nearly all microbiology labs utilize corrosive acids, alkalis, and organic compounds that are toxic. The potential for toxicity can manifest itself through amounts as small as the fumes released by opening the container. Even a whiff of a concentrated acid or other corrosive liquid can cause tissue damage to the nasopharynx. A spill of even a few hundred milliliters of an organic chemical, like phenol, on the body can be life-threatening. Phenol vapors, in excessive amounts, can cause damage to the nasopharyngeal mucosa and to the mucous membranes of the eye. Brief exposure to phenol vapors can cause minor irritation of these mucosa as well. If one uses phenol frequently, it is sensible to perform the manipulations in a chemical fume hood if possible. Sources of microbial contamination to you and others are aerosols formed by the handling of the inoculating loop, prepara- 120 Haidaris and Brownlow
tion of slides, plating of cultures, the pouring of microbial suspen sions, and pipetting. These procedures can be serious sources of infection if a hazardous pathogen is being handled. Each of these actions will be discussed individually. For individuals whose body defenses are compromised by underlying disease or medical treat ment, it is sensible for them to check with their physician as to the potential hazards to them of working in a microbiology lab where even organisms that are normally nonpathogenic are being handled The Inoculating Loop Excessively long or improperly made loops can shed their inoculum, either by vibration or spontaneously. A film formed by a loopful of broth culture that is vibrated can break the surface tension that keeps the film in place, forming an aerosol. The longer the loop is, the more vibration that ensues from handling An incompletely closed loop can also easily result in a break in surface tension of the film. The optimal size of loop is approxi mately 2 to 3 mm in diameter, and the loop should be completely closed. The length of the wire portion of the loop, or shank, should be approximately 5 to 6cm. If a large flask is being inoculated tilting the flask to bring the liquid closer to the neck may be a way to avoid the use of a very long wire. When loops become excessively bent or encrusted with carbonized material, they should be replaced. Pre-sterilized, single-use plastic loops are also available. They are not to be placed in contact with flame or solvents such as acetone and should be discarded into a disinfec tant solution The discharge of proteinaceous or liquid material that often follows flaming a loop has been suspected as a source of contam- ination, but there is little evidence to support this contention Nonetheless, it is best to decontaminate the wire loop by placing it into the apex of the internal blue flame of the burner so that any discharged material has to pass through the bulk of the flame as it leaves the loop. Preparation of slid The production of aerosols by spreading of a bacterial suspension on a slide is minimized by gentle movements, espe cially when removing the loop from the spread suspension For pathogens this activity is best performed in a biosafety hood, and the slides should not be removed from the hood until completely dried Working Safely with Biological Samples 12
tion of slides, plating of cultures, the pouring of microbial suspensions, and pipetting. These procedures can be serious sources of infection if a hazardous pathogen is being handled. Each of these actions will be discussed individually. For individuals whose body defenses are compromised by underlying disease or medical treatment, it is sensible for them to check with their physician as to the potential hazards to them of working in a microbiology lab where even organisms that are normally nonpathogenic are being handled. The Inoculating Loop Excessively long or improperly made loops can shed their inoculum, either by vibration or spontaneously. A film formed by a loopful of broth culture that is vibrated can break the surface tension that keeps the film in place, forming an aerosol. The longer the loop is, the more vibration that ensues from handling. An incompletely closed loop can also easily result in a break in surface tension of the film. The optimal size of loop is approximately 2 to 3 mm in diameter, and the loop should be completely closed.The length of the wire portion of the loop, or shank, should be approximately 5 to 6 cm. If a large flask is being inoculated, tilting the flask to bring the liquid closer to the neck may be a way to avoid the use of a very long wire. When loops become excessively bent or encrusted with carbonized material, they should be replaced. Pre-sterilized, single-use plastic loops are also available. They are not to be placed in contact with flame or solvents such as acetone, and should be discarded into a disinfectant solution. The discharge of proteinaceous or liquid material that often follows flaming a loop has been suspected as a source of contamination, but there is little evidence to support this contention. Nonetheless, it is best to decontaminate the wire loop by placing it into the apex of the internal blue flame of the burner so that any discharged material has to pass through the bulk of the flame as it leaves the loop. Preparation of Slides The production of aerosols by spreading of a bacterial suspension on a slide is minimized by gentle movements, especially when removing the loop from the spread suspension. For pathogens this activity is best performed in a biosafety hood, and the slides should not be removed from the hood until completely dried. Working Safely with Biological Samples 121
Streaking of plates In general, aerosol production is minimized by using smooth plates. Rough surfaces or bubbles in the agar can lead to exces- sive vibration of the loop. Spreading of samples with a sterile glass rod may minimize the production of aerosols on agar plates with rough surfaces Pouring of Microbial Suspensions Following centrifugation, pouring off the supernatant from a microbial suspension can result in aerosols. One safe way to min imize aerosol production is to use a funnel with the narrow end submerged into disinfectant in a large container. Use enough disinfectant to mediate the decontamination of the supernatant and a container large enough to avoid overflow. A volume of disinfectant equal to, or greater than, the amount of supernatant to be decontaminated should suffice. Rinse the funnel with more disinfectant to handle any residual supernatant clinging to th funnel wal Even for solutions deemed to be safe, it is good practice to never mouth pipette. It is mandatory not to mouth-pipette a microbial solution, even with a cotton-plugged pipette. Aspiration of organisms into the mouth can occur despite the cotton plug Blowing out the last few droplets from a pipette can also form aerosols. Use either a manual or automatic pipetting aid to pipette. The discharge of the last few droplets using either manual or automatic pipette aids can result in aerosols, so avoid this if possible. If it is necessary to discharge the entire contents of pipette, try to avoid spraying. Again, for serious pathogens, pipet ting should be performed in a biosafety hood. Contaminated pipettes should be discarded into a container containing a suffi cient volume of disinfectant to permit the complete immersion of the pipette Are You Prepared to Deal with an Emergency Adequate preparation for an emergency requires both an appreciation for the potential hazards involved and knowledge of the resources available to handle the emergency. Such ration should precede actual lab work, although this is rarely done We have discussed the appropriate types of laboratory clothing that should be worn in the microbiology laboratory, important safety equipment and supplies, and potential sources of harm 122 Haidaris and Brownlow
Streaking of Plates In general, aerosol production is minimized by using smooth plates. Rough surfaces or bubbles in the agar can lead to excessive vibration of the loop. Spreading of samples with a sterile glass rod may minimize the production of aerosols on agar plates with rough surfaces. Pouring of Microbial Suspensions Following centrifugation, pouring off the supernatant from a microbial suspension can result in aerosols. One safe way to minimize aerosol production is to use a funnel with the narrow end submerged into disinfectant in a large container. Use enough disinfectant to mediate the decontamination of the supernatant, and a container large enough to avoid overflow. A volume of disinfectant equal to, or greater than, the amount of supernatant to be decontaminated should suffice. Rinse the funnel with more disinfectant to handle any residual supernatant clinging to the funnel wall. Pipetting Even for solutions deemed to be safe, it is good practice to never mouth pipette. It is mandatory not to mouth-pipette a microbial solution, even with a cotton-plugged pipette. Aspiration of organisms into the mouth can occur despite the cotton plug. Blowing out the last few droplets from a pipette can also form aerosols. Use either a manual or automatic pipetting aid to pipette. The discharge of the last few droplets using either manual or automatic pipette aids can result in aerosols, so avoid this if possible. If it is necessary to discharge the entire contents of the pipette, try to avoid spraying. Again, for serious pathogens, pipetting should be performed in a biosafety hood. Contaminated pipettes should be discarded into a container containing a suffi- cient volume of disinfectant to permit the complete immersion of the pipette. Are You Prepared to Deal with an Emergency? Adequate preparation for an emergency requires both an appreciation for the potential hazards involved and knowledge of the resources available to handle the emergency. Such preparation should precede actual lab work, although this is rarely done. We have discussed the appropriate types of laboratory clothing that should be worn in the microbiology laboratory, important safety equipment and supplies, and potential sources of harm. 122 Haidaris and Brownlow