Protection of Biosafety Cabinet Vacuum Lines
During a number of laboratory inspections it has become clear that the vacuum line protection system for use in a biosafety cabinet (also called “tissue culture hood”, or “laminar flow hood”) is not being used correctly.
The wall vacuum system is piped throughout the building from a large vacuum pump and reservoir in one of the building’s mechanical spaces. Air drawn through the system is simply exhausted out the vent side of the pump without any treatment. Maintenance personnel frequently occupy these spaces, and it is essential that our maintenance personnel be protected from potentially harmful materials that could be accidentally introduced into the vacuum system during aspiration.
There are two sets of circumstances where the vacuum protection system is used. One is for general microbiological work, and one is for medium- to large-scale cell or tissue culture work.
General microbiological, or small-scale tissue culture, work in a biosafety cabinet
The flasks are to be kept inside the cabinet when connected to the vacuum system, per CDC requirements in “Primary Containment for Biohazards, CDC publication, 2009.
This document clearly states that “Bulky items such as biohazard bags, discard pipette trays and vacuum collection flasks should be placed to one side of the interior of the cabinet.” These items can be arranged along the back of the cabinet, or clustered in a corner near the vacuum connection. This system is also described in Appendix B of the CDC/NIH document “Biosafety in Microbiological and Biomedical Laboratories”, 5th Ed., 2009, which the UCSF Institutional Biosafety Committee has adopted in its entirety as a governing document for describing biosafety practices.
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The proper system is comprised of two sidearm vacuum flasks in series, connected to a HEPA (not membrane) filter, and then to the hose bib on the side of the cabinet. Some HEPA filters, unlike membrane filters, are autoclavable and reusable, and there are disposable units available also. A non-disposable HEPA filter should be washed and autoclaved any time it becomes contaminated or plugged, otherwise it should be cleaned periodically. Recommended cleaning frequencies are every six months if the vacuum system is used frequently, or annually if the vacuum system is used infrequently.
As long as at least 30 minutes has passed since the last potentially biohazardous liquid has been aspirated into the flask, the solution in the flask may be discarded down the sink.
The flasks should be emptied and fresh disinfectant added periodically depending on how frequently they are used. If the system is used more than twice a week, the flasks should be emptied, washed and re-filled weekly. If used between twice a week and twice a month, they should be cleaned monthly. If used rarely, the flasks should be emptied, washed after each use, and the flasks should be refilled with fresh bleach just before use.
Medium- to large-scale tissue or cell culture work in a biosafety cabinet
The system requirement for flasks kept in the cabinets pre-dates the extensive use of filter flask systems for extracting high volumes of potentially contaminated cell culture media. Keeping the flasks inside the cabinet requires the use of relatively small flasks that would need to be emptied frequently, necessitating subsequent re-sterilization of the cabinet interior. The principal hazards introduced by keeping the flasks outside of the cabinet, especially on the floor, are that the cabinet sash cannot be fully closed because of the tubing, which prevents the cabinet from completely sterilizing; and it is possible that, if kept on the floor, one or both of the flasks could become disconnected from the vacuum line and spill, or be knocked over and broken.
In order to mitigate these potential hazards, the following requirements must be met:
a) If you need to keep the two-flask system outside of the biosafety cabinet, you must place both flasks in a secondary container that is large enough to hold the volume of both flasks.
b) After potentially contaminated media has been drawn off, draw enough of a 1:10 dilution of standard household bleach to continuously fill the tubing.
For both systems
- The potentially contaminated flask contents must be in contact with bleach solution for at least 30 minutes before being disposed down the drain.
- When returning the empty flasks to service, fill them each with 10% of their volume with undiluted bleach. For example, a four liter flask would have 400 ml of undiluted bleach added before being reconnected to the vacuum system.
- Turn off the vacuum when it is not being used. This saves energy at the pump and helps keep the system clean.
If you have questions about this policy, please contact your Environment, Health and Safety Specialist or call OEH&S at 476-1300 and ask for one of the Biosafety Officers.
Suggested Equipment List for Vacuum Systems
Sidearm vacuum flasks should be clear, not translucent. Use flasks such as the ones described below or similar. Because they are a contract vendor for UCSF, Fisher Scientific products are described. These are not the only items available, similar items may be used from other sources as long as general quality and durability are not compromised.
From Fisher Scientific:
Fisherbrand* Reusable Heavy-Wall Filter Flasks
Fisher Scientific Catalog #: FB-300-xxx, where xxx is the volume in ml. from 250 to 4000.
Whatman* HEPA-Vent* Filter Inlet/Outlet: 1/4 to 3/8 in. tapered hose barb
Fisher Scientific Catalog #: 09-744-79
Whatman* HEPA-Cap* Disposable Air Filtration Capsules
Fisher Scientific Catalog #: 09-744-12
Note: the Whatman* Vacu-Guard* Filter (Fisher Scientific Catalog #: 09-744-75) is not a HEPA filter, and should not be used.