The use of blenders, ultrasonic disrupters, grinders, and lyophilizers can result in considerable aerosol production. This equipment, and any other device that may generate an aerosol, must be used in a BSC when working at BSL-2 or BSL-3, or may require assessment for the use of respiratory protection
Safety blenders are designed to prevent leakage from the bottom of the blender jar, provide a cooling jacket to avoid biological inactivation and to withstand sterilization by autoclaving. Test blender rotors with sterile saline or dye solution to determine if they are leak-proof prior to use with recombinant and/or biohazardous material. The use of glass blender jars is not recommended because of the breakage potential. If they must be used, glass jars must be covered with a polypropylene jar to prevent spraying of glass and contents in the event the blender jar breaks. A towel moistened with disinfectant must be placed over the top of the blender during use. Before opening the blender jar, allow the unit to rest for at least one minute to allow the aerosol to settle and then open in a BSC. The entire device and jar must be decontaminated promptly after use.
Sonicators and French Presses
Sonication of living microorganisms is potentially a source of aerosols. Whether using a sonicating bath or probe sonicator, precautions must be taken to protect personnel. Ordinarily, this will be done by performing the sonication in a BSC. It is prudent to consider all surfaces in the vicinity of the sonicator to be contaminated following its use, and they must be thoroughly disinfected. Modern sonicators have containment mechanisms that must be utilized if available.
The use of French presses requires similar caution. The greatest potential for aerosols is at, or near, the end of a pressing cycle when air bubbles at the top of the column of suspension can escape with little or no warning. This can result in microaerosols, which will contaminate the work area, but also in macroaerosols which can effectively inoculate the mucus membranes and conjunctivae of the operator. Due to the size of the press, it is usually impractical to perform this operation inside a BSC. The pressing of human pathogens outside of a BSC must be approved by RLSS, and operators must use face shields or other means of eye protection.
Arcing, which sometimes occurs during electroporation of bacteria, can also cause aerosols. These range from minimal spattering of the bacteria/DNA solution to major broadcast of potentially infectious material when a cuvette shatters. The shields on most electroporators are usually sufficient to protect the operator from flying plastic and gross contamination, but will not contain microaerosols. Thus, if one must electroporate bacteria at BSL-2 or BSL-3, it must be done in a BSC.
Cell concentrators are also employed in laboratories as a means of handling viable organisms. There are two principal types of cell concentrators. The first involves the removal (through evaporation) of liquid from solid material thereby increasing the concentration versus volume. The second involves the retention of the solid material on the surface of a filter and the subsequent harvesting of the material from the filter surface. The following safety rules must be applied when using such an apparatus:
- Before starting, check all of the equipment to be used for signs of stress or fatigue. Pay close attention to tubing and glassware.
- When possible conduct the procedure in a BSC.
- Upon the completion of the run, thoroughly sanitize the apparatus before the next experiment.
- For rotary type concentrators, make sure the load is balanced.
- If a vacuum is to be used, make sure the appropriate exhaust filter is present on the vacuum line to prevent contamination.
Do not exceed recommended pressures or speed for operation of equipment.
Lyophilizers and Ampoules
Depending on lyophilizer design, aerosol production may occur when material is loaded or removed from the lyophilizer unit. If working at BSL-2 or BSL-3, sample material must be loaded in a BSC. The vacuum pump exhaust must be filtered to remove any hazardous agents or, alternatively, the pump can be vented into a BSC. After lyophilization is completed, all surfaces of the unit that have been exposed to the agent must be disinfected. If the lyophilizer is equipped with a removable chamber, it must be closed off and moved to a BSC for unloading and decontamination. Handling of cultures must be minimized and vapor traps must be used.
Opening ampoules containing liquid or lyophilized culture material at BSL-2 and BSL-3 must be performed in a BSC to control the aerosol produced. To open, nick the neck of the ampoule with a file, wrap it in a disinfectant soaked towel, hold the ampoule upright and snap it open at the nick. Reconstitute the contents of the ampoule by slowly adding liquid to avoid aerosolization of the dried material. Mix the contents without bubbling and withdraw it into a fresh container. Discard the towel, ampoule top, and bottom as biohazardous material waste.
Ampoules used to store biohazardous material in liquid nitrogen have exploded, causing eye injuries. The use of polypropylene tubes eliminates this hazard. These tubes are available dust-free and pre-sterilized, and are fitted with polyethylene caps with silicone washers. Heat sealable polypropylene tubes are also available.