Biological Safety

Biosafety Guide

 

Biohazardous Sharps Disposal

  1. Seal the sharps container lid, apply heat sensitive tape to the container, and autoclave for a minimum time of 30 minutes at 121°C and 15psi
  2. Label the container “autoclaved”
  3. Arrange for container pick up: 
  • Submit a Hazardous Materials Waste Pickup Request and note on the request that the material has been autoclaved, or
  • Leave your autoclaved sharps container in the designated accumulation area in Bio II 4106, LSB 2204, or LSB 4218, where it will be picked up without a request.

Non-biohazardous laboratory sharps disposal procedures: https://www.ehs.ucsb.edu/labsafety-chp/sec2/laboratory-sharps-disposal

Biosafety Cabinet Use

  • The laminar airflow is based on the sash height and airflow through the front and back grills. For this reason, keep elbows, forearms, notes, equipment, etc., clear of the front and rear grills.
  • The intake air velocity past the sash at working height is only 100-150 linear feet per minute. It’s important to minimize foot traffic past the BSC when it’s in use; walking past a BSC is enough to disrupt the laminar air flow.
  • Move your hands straight in and out when necessary. Place a waste container in the back to one side to reduce the number of times that you take your hands in and out.
  • Work 4-6 inches inside the BSC where the laminar airflow is at its most uniform.
  • Divide the work space into clean and contaminated sides, and make sure that work flows from the clean area towards the contaminated area.
     

Biological safety cabinets must be tested and certified for proper function after installation, relocation, or maintenance, and at least annually. The University of California has negotiated a system-wide agreement with Technical Safety Services, Inc., outlining service expectations and pricing schedules. The Los Angeles branch serves UCSB and a field manager may be reached at 562-694-3626. Lead times vary, but technicians can usually schedule services within a week.

Biosafety Level 2 Containment Work Practices

Hand Hygiene

  • Wash hands upon contact with anything potentially infectious, after work, after removing gloves and before leaving the laboratory
  • Gloves are replaced as soon as practical when contaminated, torn, punctured, or when their ability to function as a barrier is compromised

Aerosol Containment

  • Perform all procedures in a manner that minimizes splashes, the creation of droplets or aerosols
  • Perform procedures that may generate infectious aerosols in a biosafety cabinet
  • Use a mechanical pipetting devices at all times

Universal Precautions

  • Universal precautions is an approach to infection control in which you treat all human and nonhuman primate blood, tissues and body fluids as if they are potentially infectious for HIV, hepatitis B virus, and other bloodborne pathogens, (Bloodborne Pathogens Standard 29 CFR 1910.1030(b) definitions).
  • Implementing universal precautions means that you consistently adhere to
    • Hand hygiene
    • Wearing non-porous personal protective equipment as a physical barrier between you and the potentially infectious material
    • Safe work practices specific to sharps
    • Routine surface disinfection
    • Waste deactivation and disposal

Delineation of Laboratory from Non-laboratory Operations

  • Entry ways to work areas, containers of biohazardous waste, refrigerators and freezers used with potentially infectious materials must have labels with the word “BIOHAZARD” and the universal biohazard symbol in orange-red or red with lettering and symbols in a contrasting color.
  • Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in work areas
  • Food and drink must not be stored in laboratory refrigerators, freezers, shelves, cabinets, or bench tops that contain research material(s)
  • Transport biological agents between laboratories and campus buildings using rigid, leak-proof, double container systems that are lined with enough absorbent material to absorb liquid leaks

Sharps Safety

  • Do not bend, break, shear or remove needles from disposable syringes
  • Dispose of contaminated sharps in a single-use, unlined, disposable container that is rigid, leak proof, puncture resistant, and labeled with the biohazard symbol and “SHARPS WASTE”; close the container when it is not in use
  • Place sharps waste containers as near the point of use as appropriate for immediate disposal and do not exceed the sharps container fill line at 2/3 full

Disinfection

  • Disinfect the work area and lab equipment daily and after use
  • Use an EPA-registered, high level disinfectant for work with potential sources of bloodborne pathogens

Written Procedures

  • Standard Operating Procedures should be established for techniques, e.g., cell culturing or tissue homogenization, and equipment, e.g., fluorescence activated cell sorters or sonicators
  • Methods to prevent occupational exposures are specified by the Principal Investigator within the Biological Use Authorization

Human Blood, Tissues and Cells

Biosafety level 2 containment practices are used for work with any human and non-human primate blood, body fluids, tissues, or primary cells.
The biological use authorization should describe what is known about these materials, including

  • Cell or tissue types
  • Source, i.e., hospital or clinic, tissue bank, collaborator, or commercial vendor
  • Whether the materials are known to be infectious for a certain disease indication
  • Whether the materials have been screened for common bloodborne pathogens, and which pathogens are included in the panel. Tests are performed on batches and results are tracked by the lot number. Results, when available, are provided for the specific lot on the Certificate of Analysis, showing either a "negative" or "positive" specification or result for the assay.

Selected human tissue vendors with their bloodborne pathogens screening panel:

Addex Bio Technologies: HIV, HBV, HCV, HPV, EBV, CMV
ATCC: HIV, HBV, HCV, HPV, EBV, and CMV
Bioreclamation IVT: HIV, HBV, HCV, West Nile Virus, Trypanosoma cruzi and serologic tests for syphilis
Cedarlane: Not screened
CHTN: Not screened
DSMZ:  HIV, HBV, HCV, EBV, HHV-8, HTLV-1/2, MLV, SMRV
EMD Millipore: HIV, HBV, HCV, EBV, HAV, HSV
ScienCell: HIV, HBV, HCV
Thermo Fisher Scientific: HIV, HBV, HCV

Liquid Waste Collection in a Biological Safety Cabinet

Protect the laboratory vacuum system from contamination with a second flask and an inline HEPA filter. The primary flask contains full strength bleach and is used to collect spent media. The second flask collects any overflow. A HEPA filter is adjacent to the vacuum port for added protection. If one or both flasks are kept on the floor, they must be in a plastic container to prevent breakage.

Medical Waste Procedures

Solid waste is collected in red biohazard waste bags in secondary containment, treated at a minimum of 121°C for at least 30 minutes in an autoclave permitted with CDPH, and disposed of in the red-lidded totes designated for laboratory waste. Do not retain solid medical waste for longer than 7 days at room temperature. 

Liquid waste is treated with an EPA-approved, tuberculocidal disinfectant and then dispensed down the laboratory sink with an excess of water. Tissue culture liquid waste is typically deactivated by aspirating it into 100% bleach. Add spent media until the bleach is diluted 10-fold to 10% final vol/vol, allow to set for a minimum of 20 minutes, and dispense down the laboratory sink with an excess of water. Do not retain liquid medical waste for longer than 7 days at room temperature.  

Trace chemotherapeutic and pharmaceutical waste, other than RCRA hazardous waste or Controlled Substances, is disposed of through EH&S as chemical hazardous waste. Residual solids and contaminated items (including sharps) are collected in a rigid, lidded container labeled with "CHEMO" on the Hazardous Waste Container label. Chemical hazardous waste must be picked up by EH&S Hazardous Waste within 270 days of the accumulation start date. 

Biohazardous/medical sharps disposal procedures are described above, and the complete campus medical waste management plan is available on the Medical Waste tab.

Noninfectious Biological Waste

Not all biological waste is biohazardous, or infectious, medical waste, although it must be treated to deactivate cell cultures, genetically modified organisms, recombinant nucleic acids, etc.
"Biotechnological" or noninfectious biological solid waste is collected in clear/white autoclave bags. 
Examples of clear/white autoclave bag solid waste: vials containing transgenic fruit flies, plates of recombinant S. cerevisiae or E. coli K12, and well-characterized cell lines. 

Personal Protective Equipment

  • Long pants and closed toe shoes are required for entering and occupying a laboratory.
  • While working with, or being adjacent to hazardous materials or processes, an appropriate lab coat and safety eyewear are also required.
  • A lab coat and disposable latex or nitrile gloves are worn when working at a biological safety cabinet.
  • Safety glasses or goggles, with an accompanying face shield for handling large volumes, are worn for work outside of a biological safety cabinet.
  • Barrier lab coats that are impervious to small volumes of liquids are used for work with human blood or other infectious liquid handling procedures.
  • In case of gross contamination with infectious biological materials, lab coats are to be bagged and autoclaved prior to laundering via Mission Linen.

Post Exposure Procedures

  1. Clean the affected area.  Wash needle sticks and cuts with soap and water, flush splashes to the nose, mouth, or skin with water, and irrigate eyes with clean water or sterile saline. The laboratory sink, emergency shower and eye wash stations will be used as necessary to flush affected areas with water for several minutes.
  2. Report the incident. Exposures must be reported to the PI or Lab Safety Contact immediately. Staff exposures or injuries are recorded with an Employer’s First Report: https://www.ehs.ucsb.edu/workcomp Student exposures or injuries are filed via this webpage: https://www.ehs.ucsb.edu/riskmanagement/3rd-party-incidents
  3. Seek treatment. Healthcare personnel treating exposed patients must be informed of the biological materials involved in the exposure. Employees and personnel on University pay status seek treatment at Sansum Occupational Medicine Center, 101 South Patterson Avenue, Santa Barbara, CA 93111, and after business hours at Sansum Urgent Care Center, 215 Pesetas Lane, Santa Barbara, CA 93110. Students are to seek treatment at UCSB Student Health Services, Building 588, at El Colegio and Ocean Road, Santa Barbara CA 93117, and after business hours at Goleta Valley Cottage Hospital, 351 S. Patterson Avenue, Goleta CA 93117.

Relocation Guide for Biological Safety

Packing and Transporting Biological Materials across Campus

  • Use a rigid, leak-proof, double container system
  • Containers must be readily decontaminated, i.e., not Styrofoam or cardboard
  • Seal both the inner and outer containers with Parafilm
  • Pack items to prevent breakage or leakage
  • Line the outer container with enough absorbent material to soak up the volume of liquid
  • Do not leave packages unattended
  • Label the container with the biohazard symbol and the PI/designee contact information
  • Decontaminate the outside of the primary and secondary containers, including any handles, before you exit the lab, so that you can safely doff PPE before exiting
  • Bring clean disposable gloves to unpack the container, and carry your lab coat in an unused autoclave bag

Steps to Relocating a Biosafety Cabinet

  1. Authorized personnel/researchers empty the cabinet and decontaminate interior work areas
  2. An outside vendor, Technical Safety Services, Inc., performs a decontamination procedure on the entire unit
  3. UCSB Furniture Services moves the cabinet to the new location, or into surplus
  4. The PI’s departmental staff finalizes installation 
  5. Technical Safety Services, Inc. recertifies cabinet performance

Spill Cleanup

For spills contained within a biosafety cabinet, keep the cabinet blower on. 

  1. Replace any contaminated personal protective equipment. 
  2. Obtain or prepare a fresh solution of disinfectant. 
  3. Cover the spill with paper towels to prevent aerosols and splashing, and apply disinfectant to the area.
  4. Wait out 10 minutes. 
  5. Use paper towels to absorb the spill, working from the outside in; use tongs to collect the paper towels if sharps are involved.
  6. Bag the clean-up materials and disposable gloves as solid waste, then wash your hands thoroughly.

For larger spills outside of a biosafety cabinet, notify colleagues and vacate the premises for 30 minutes to allow time for aerosols to settle and for a few room air changes.  
Post a sign at the door warning of the spill and advising of the re-entry time. 
Before or upon re-entry, put on clean personal protective equipment and proceed as described above.

Viral Vectors in Tissue Culture and in Animals

Information for Viral Vector Characterization and Risk Assessment
Vector System

  • Vector type, e.g., lentiviral, adenoviral, pseudorabies, adeno-associated, etc.
  • Host cell range i.e., vector tropism or which cell types may the vector infect
  • Replicating or non-replicating; design features rendering the viral vector replication incompetent
  • Number of plasmids used in the system
  • Promotor type and whether it is inducible
  • Any design features rendering the viral vector safer to the researcher

Transgene or Insert Information

  • Name and function of the transgene or insert. Specify whether the insert is oncogenic, immunoregulatory, metabolic, a fluorescent reporter, etc. Special care should be given to the design and handling of virus vectors containing genes that make growth-regulating products, products released into the circulation, and products that may have a general effect on the host/researcher immune system.
  • Source organism of the gene or insert, e.g., human, rodent, etc.

Experimental Procedures

  • Source or site of generation, e.g., laboratory, core facility, or commercial provider
  • Is there a dilution or concentration step, or is the supernatant from the packaging cell line used?
  • The vector titer and total amount of vector 
  • The volumes of viral particles that will be handled; containment practices may be modified for larger volumes
  • Site of inoculation
  • Method of administration
  • How or whether the vector is expected to be shed

Work with Biological Toxins

  • Experiments should be planned to eliminate or minimize work with dry, powdered toxin. Dry toxin must be manipulated using containment equipment such as a disposable glove bag, chemical fume hood or a biological safety cabinet.
  • Evaluate and modify experimental procedures to reduce or eliminate the possibility of aerosolizing solutions containing toxin
  • For complex operations, new workers may want to perform practice runs in which the procedures are rehearsed without active toxin
  • Work with toxins should only be done only in laboratories with controlled access and at pre-determined bench areas. When toxins are in use, the room should be clearly posted: “Toxins in Use—Authorized Personnel Only.”
  • Containers used for toxin storage should be sealed, labeled, and secured to ensure restricted access
  • Consideration should be given to requiring the presence of at least two knowledgeable individuals at all times in the laboratory for high-risk operations, e.g., manipulations with dry forms of toxins, intentional aerosol formation, and the use of hollow-bore needles in conjunction with amounts of toxin estimated to be lethal for humans

Research groups that uses toxins must augment the laboratory Chemical Hygiene Plan with a Standard Operating Procedure (SOP) for the toxin or class of toxins. The researcher should be sufficiently adept at the planned experimental procedures before working with the toxin.

SOP Components:

  • Procurement
  • Storage location and security
  • Inventory/tracking
  • Areas designated for work
  • Glove choice based on how the toxin is solubilized (aqueous buffer or organic solvent)
  • Methods to transfer liquids containing toxin
  • Deactivation methods
  • Routine decontamination of laboratory surfaces and equipment
  • Solid and liquid waste disposal procedures
  • Spill cleanup procedures