Biological Safety Manual

B4.1

B4.2

B4.3

B5 BIOCONTAINMENT

Four basic levels of biocontainment, or biosafety levels (BSLs), are defined in both the BMBL
(pg.27) and the NIH Guidelines (Section II-B, referred to as BLs). Only BSLs pertinent to University
research are discussed below. The levels build upon each other in ascending order by degree of
protection provided to personnel, the environment, and the community such that the requirements
for BSL2 are those for BSL1 with additional, defined protection measures. Each level consists of
combinations of laboratory practices, safety equipment, and laboratory facilities which provide
containment to allow manipulation of biological agents of increasing danger to life, health, and/or
the environment. As the science and discipline of biosafety has evolved biocontainment
designations for animal (ABSL), plant (PCL), and arthropod (ACL) have been better defined.

B5.1

B5.2

Animal Biocontainment
Four biosafety levels are also described in the BMBL and NIH Guidelines for activities involving
infectious disease work with vertebrates - Animal Biosafety Levels 1, 2, 3, and 4, (ABSL1, ABSL2,
ABSL3, ABSL4). As with general biosafety and containment, they provide increasing levels of
protection to personnel and the environment. Only ABSL1 and ABSL2 are described below. In
addition to ABSL levels described by the BMBL, the United States Department of Agriculture
(USDA) has developed facility parameters and work practices for handling agricultural pathogens,
ABSL-2Ag through ABSL-4Ag. See Appendix D of the BMBL for additional information.
Animal work can present unique hazards not found in standard biological laboratories. The coapplication of BSL and ABSL are determined by a protocol-specific risk assessment.
Work involving animals in conjunction with potentially infectious materials or work
involving animal pathogens must be approved by both IACUC and the IBC; IBC approval
should be sought first as it is likely required for IACUC approval.
Please see the IACUC website for additional details and IACUC protocol submission forms. The
checklists for the CDC import permit program are helpful tools in assessing animal containment.
Harmonized checklists from the BMBL are in sections B9.2.3.1 and B9.2.3.2.

B5.3

Arthropod Biocontainment
Resources used as guidance for arthropod biosafety, biosecurity, and biocontainment:
Arthropod Containment Guidelines (currently version 3.2) as published by the American Committee
of Medical Entomology and the American Society of Tropical Medicine and Hygiene.
USDA-PHIS Containment Guidelines for nonindigenous phytophagous arthropods and their
parasitoids and predators.
CDC Import Permit checklists for arthropod containment.
These resources have been used to develop harmonized checklists for research involving housing
arthropods. Checklists for ACL1 and ACL2 are in sections B9.2.3.3 and B9.2.3.4, respectfully.

B5.4

Plant Biocontainment
Resources used as guidance for plant biosafety, biosecurity and biocontainment:
A Practical Guide to Containment: Plant Biosafety in Research Greenhouses
USDA-APHIS Containment Guidelines for the following: 1) Plant Pathogenic Bacteria, 2) Fungal
Plant Pathogens, 3) Viral Plant Pathogens and their Vectors, 4) Pathogenic Nematodes, and 5)
Noxious Weeds and Parasitic Seed Plants.
These resources in addition to the NIH Guidelines have been used to develop harmonized
checklists for research involving plants. Checklists for PCL1 and PCL2 are in sections B9.2.3.5
and B9.2.3.6.

B6 CLASSIFICATION OF BIOLOGICAL MATERIALS

B6.1

Introduction
The NIH and the CDC publish containment measures for work with infectious microorganisms.
These different categories of biological containment are addressed in Section B5 above.
Classification of an organism into a risk group involves evaluation of multiple factors. Information
for certain agents can be found in Appendix B of the NIH guidelines and in the Agent Summary
Statements in the BMBL. The categorization of biological agents into risk groups (RGs) is done
according to the hazard they pose to personnel and the environment.
As indicated in B4.3.4, the IBC determines the containment level and classification of agents at
TTU. “No one should conclude that the absence of an agent summary statement for a human
pathogen means that the agent is safe to handle at BSL-1 or without a risk assessment to
determine the appropriate level of containment.” (BMBL 6th ed. pg 5)

B6.2

B6.3

Genetically Engineered/Modified Organisms (GMOs/GMMOs)
All work with genetically engineered organisms is to be done in compliance with the NIH
Guidelines. These guidelines classify recombinant/synthetic nucleic acid experiments into four risk
groups as discussed in the previous section. The USDA requires permits for field testing of
genetically engineered plants. A summary (current as of April 2019) is below; please refer to the
NIH Office of Science Policy (NIH-OSP) website for the most current guidelines. Certain
experiments require approval by the NIH-OSP in addition to the TTU IBC.
B6.3.1
Experiments covered by the NIH Guidelines per Section III
All Experiments involving rDNA molecules require registration with the IBC regardless of
NIH exemption per Section III-E or -F.
B6.3.1.1
Experiments that require NIH Director Approval and IBC approval before initiation (Section
III-A) include those:
(a) Experiments involving the deliberate transfer of a drug resistance trait to microorganisms
that are not known to acquire the trait naturally, if such acquisition could compromise the
ability to control disease agents in humans, veterinary medicine, or agriculture.
B6.3.1.2
Experiments That Require NIH OSP and Institutional Biosafety Committee Approval Before
Initiation (Section III-B):
(a) Experiments involving the cloning of toxin molecules with LD50 of less than 100 nanograms
per kilogram body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin,
diphtheria toxin, and Shigella dysenteriae neurotoxin).
B6.3.1.3
Experiments Involving Human Gene Transfer that Require Institutional Biosafety Committee
Approval Prior to Initiation (Section III-C):
(a) Experiments involving the deliberate transfer of recombinant or synthetic nucleic acid
molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules,
into one or more human research participants.
B6.3.1.4
Experiments that Require Institutional Biosafety Committee Approval Before Initiation
(Section III-D):
(a) Experiments that use Risk Group 2, 3, 4, or restricted agents as host-vector systems;
(b) Experiments in which DNA from risk group 2, 3, 4, or restricted agents is cloned into
nonpathogenic, prokaryotic, or lower eukaryotic host-vector systems;
(c) Experiments that involve the use of infectious DNA/RNA virus, or defective DNA/RNA virus
in the presence of helper virus in tissue culture systems;
(d) Experiments that involve whole plants or animals;
(e) Experiments that involve more than 10 liters of culture; and/or
(f) Experiments that involve influenza viruses.
B6.3.1.5
Experiments that must be registered at the time of initiation include those (Section III-E):
(a) Involving the formation of recombinant or synthetic nucleic acid molecules containing no
more than 2/3 of the genome of any eukaryotic virus;
(b) Involving nucleic acid molecule-modified whole plants, and/or experiments involving
recombinant or synthetic nucleic acid molecule-modified organisms associated with whole
plants, except those that fall under Section III-A, III-B, III-D, or III-F of the Guidelines.
(c) Biosafety level 1 experiments involving the generation of transgenic rodents in which the
animal’s genome has been altered by stable introduction of recombinant or synthetic nucleic
acid molecules, or nucleic acids derived therefrom, into the germline.
B6.3.1.6
Experiments exempt from the NIH Guidelines that require registration with the IBC but may
be initiated immediately (Section III-F):
(a) Those synthetic nucleic acids that: (1) can neither replicate nor generate nucleic acids that
can replicate in any living cell, and (2) are not designed to integrate into DNA, and (3) do
not produce a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per
kilogram of body weight.
(b) Recombinant or synthetic nucleic acid molecules that are not in organisms, cells or viruses
and that have not been modified/manipulated to render them capable of penetrating cellular
membranes.
(c) Recombinant or synthetic nucleic acid molecules that consist solely of the exact
recombinant or synthetic nucleic acid sequence from a single source that exists
contemporaneously in nature.
(d) Those that consist entirely of nucleic acids from a prokaryotic host including its indigenous
plasmids or viruses when propagated only in that host (or a closely related strain of the
same species), or when transferred to another host by well-established physiological
means.
(e) Those that consist entirely of nucleic acids from an eukaryotic host including its
chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that
host (or a closely related strain of the same species).
(f) Those that consist entirely of DNA segments from different species that exchange DNA by
known physiological processes, though one or more of the segments may be a synthetic
equivalent. See NIH Guidelines, Appendices A-I through A-VI, for a list of natural
exchangers that are exempt from the NIH Guidelines.
(g) Those genomic DNA molecules that have acquired a transposable element, provided the
transposable element does not contain any recombinant and/or synthetic DNA.
(h) Those that do not present a significant risk to health or the environment as determined by
the NIH Director following appropriate notice and opportunity for public comment.
i. Recombinant or synthetic nucleic acid molecules containing less than one-half of
any eukaryotic viral genome (all viruses from a single family being considered
identical), that are propagated and maintained in cell in tissue culture unless they
are subject to the guidelines in Section III-B, -C, or -D.
ii. Use the following host-vector systems:
• E. coli K12 (with the exception of those experiments listed in Appendix C-II-A of
the NIH Guidelines);
• Saccharomyces cerevisiae and Saccharomyces uvarum (with the exception of
experiments listed in Appendix C-III-A of the NIH Guidelines);
• any asporogenic Bacillus subtilis and Bacillus licheniformis (which does not
revert to a spore-former with a frequency greater than 10-7 may be used for
cloning DNA (with the exception of those experiments listed in Appendix C-V-A
of the NIH Guidelines); and
• Kluyveromyces lactis, provided laboratory-adapted strains are used, (with the
exception of experiments listed in Appendix C-IV-A of the NIH Guidelines),
(i) Recombinant or synthetic nucleic acid molecules derived entirely from extrachromosomal
elements of the organisms listed in Appendix C-VI of the NIH Guidelines (including shuttle
vectors constructed from vectors described in Appendix C), propagated and maintained in
organisms listed Appendix C-VI of the NIH Guidelines (with the exception of experiments
listed in Appendix C-VI-A of the NIH Guidelines).
(j) Involve purchase or transfer of transgenic rodents for experiments at BSL1 according to
Appendix G-III-M of the NIH Guidelines.
(k) The generation of certain BSL1 transgenic rodents via breeding per Appendix C-VIII of the
NIH Guidelines.
B6.3.1.6(l)
While considered “exempt,” the BMBL guidelines still apply to the above-listed
experiments. The Biosafety Officer (BSO) a proxy, and/or IBC Chair will review the
application form and confirm that the work is classified correctly according to the NIH
Guidelines.

B6.4

Human and Non-Human Primate Materials
B6.4.1
Regulations
Work with human material is regulated by the Occupational Safety and Health Administration
(OSHA) Bloodborne Pathogens Standard, 29 CFR, Part 1910.1030. The University uses this
standard in conjunction with the BMBL and TTU OP 60.24 to provide a safe working environment
for TTU personnel.
Research involving the collection of samples from human subjects likely requires both IBC and IRB
approval.
B6.4.2
Training Requirements, Medical Surveillance, and IBC Review
B6.4.2.1
Biosafety and Bloodborne Pathogen training are required for faculty who oversee and
personnel who work in environments were human and or non-human primate material is
handled.
B6.4.2.2
Individuals who may contact human or non-human primate materials must be enrolled in
the OHP and offered the Hepatitis B vaccine series. This serves as the documented offering of
the vaccine; the OHP will handle any necessary medical surveillance. The extent of in-laboratory
medical surveillance should be discussed between the PI & EHS. Consult EHS if you have
questions regarding vaccination or medical surveillance. These measures will be documented on
the IBC protocol.
B6.4.2.3
Work with human and/or non-human primate materials requires a certified BSL2 laboratory
space and IBC protocol review. In accordance with the BMBL, all human and /or non-human
primate material should be presumed infectious and handled using BSL2 work practices and
containment; this concept is called “Universal Precautions”.
B6.4.2.4
The use of 10-20% household bleach or a hospital-grade disinfectant, registered with the
EPA for effectivity against, common bloodborne pathogens (HIV, HEP-B, HEP-C, and
Mycobacterium tuberculosis), is required for areas where human/non-human primate
materials are handled.
B6.4.3
Human and non-human primate material includes the following:
1. All fluids and excretions (blood, blood components, urine, saliva, semen, vaginal secretions,
cerebrospinal fluid, excrement, synovial fluid, sweat, etc.),
2. Unfixed tissues,
3. Fixed or unfixed brain tissue,
4. Cells (including established cell lines for cell culture), and
5. Any other material of human or non-human primate origin (apart from hair and nails).
Please contact EHS at 806-742-3876 if you have questions regarding a specific material or project.

B6.5

Biologically Derived Toxins
The BMBL requires that all biologically derived toxins be handled at BSL2 or greater containment.
Powdered forms of toxins should be replaced with toxins in suspension when possible and always
handled within containment (BSC or fume hood) using appropriate PPE to address the toxin.
Specific handling, decontamination, and waste procedures will be specified for the toxins used in
the IBC protocol.
B6.5.1
BSL2 is the minimum-required biosafety level for research involving biologically derived
toxins. Depending on the toxin and quantity, BSL3 containment may be required.
B6.5.2
In all cases of biologically derived toxin-related research, and IBC protocol is required.

B6.6

Prions and Prion-like Proteins
Prions and prion-like proteins are resistant to inactivation by heat and chemicals. Prions are
efficiently transmitted within species but can cross species barriers with reduced efficiency.
Presently, scrapie and chronic wasting disease are not known to infect humans. Prions are
transmissible by inoculation, ingestion, or transplantation of infected tissues or homogenates. Prion
infectivity is high in the brain and other central nervous system tissues and lower in lymphoid
tissues including the spleen, lymph node, gut, bone marrow, blood, and some special tissues
(placenta); at this time, aerosols do not appear to be a natural route of transmission but cannot be
excluded in a lab setting.
B6.6.1
BSL2 is the minimum-required biosafety level for research involving prions or prion-like
proteins. Depending on the nature of the research, BSL3 containment may be required.
B6.6.2
In all cases of prion-related research, including sample screening, an IBC protocol is
required.
B6.6.2.1
Principal investigators may request a waiver from the IBC protocol requirement for sample
screening purposes. Contact the IBC at ibc.ehs@ttu.edu regarding a waiver.

B6.7

Respiratory Viruses
Work with agents transmitted through respiratory exposure is a serious laboratory hazard. When
work involves the use of animals, the hazardous characteristics of zoonotic agents require careful
consideration when completing the biological risk assessment. (BMBL Section II – Biological Risk
Assessment, pg 13)
Specific considerations for Influenza Viruses
New subtypes can result from reassortment of human, swine, and avian influenza A virus genes.
Influenza A viruses of different antigenic subtypes occur naturally in many domestic and wild avian
species and have formed sustained lineages in swine, equine, and canine species. Avian origin
influenza A viruses also sporadically infect multiple other mammalian species. Interspecies
transmission and reassortment of influenza A viruses have been reported to occur among humans,
pigs, and wild and domestic fowl. The influenza A viruses responsible for the 1918, 1957, 1968,
and 2009 pandemics contained gene segments closely related to those of avian or swine influenza
A viruses.
When considering the biocontainment level and practices and procedures for work with influenza
recombinant or reassortant viruses, the IBC, will consider but not limit consideration to the
following when conducting the protocol-driven risk assessment:
• The gene constellation used;
• Any mutations that are introduced and may result in enhancement of a pathogen’s
transmissibility and/or virulence;
• Clear evidence of reduced virus replication in the respiratory tract of appropriate animal
models, compared with the level of replication of the wild-type parent virus from which it was
derived;
• Evidence of clonal purity and phenotypic stability; and
• The number of years since a virus that was antigenically related to the donor of the
hemagglutinin and neuraminidase genes last circulated.
If adequate risk assessment data are not available, a more cautious approach to containment,
utilizing elevated Biosafety Levels and practices, is warranted.
(BMBL Section VIII-E: Viral Agents, pg 259-263)
Sample screening is subject to B6.2.2 which states, “Initial processing of environmental or
“unknown” samples, specimens, and/or identification of isolates shall be done at BSL2 containment
unless additional information suggests the presence of an agent of higher risk (thus requiring BSL3
containment) or permission for use of BSL1 containment procedures and practices has been
granted by the IBC.
B6.7.1
Propagation of risk group 2 respiratory viruses of human origin or influenza viruses of any
origin requires, at minimum, the use of the BSL2-enhanced designation which includes the
following protective equipment measures:
1. Inclusion in the EHS Occupational Health Program and laboratory surveillance policies such
as fever/symptom watch, and receipt of the seasonal influenza shot.
2. Inclusion in the EHS-OHP respiratory protection program and use of respiratory protection
(N-95 or equivalent),
3. Use of either a disposable, barrier-style lab coat or a reusable, barrier-style lab coat that is
exclusive to use during respiratory viral work, and
4. Double gloving, including the taping of the inner glove over the cuff of the barrier coat so
that the outer glove can easily be changed as needed.
B6.7.3
Work stipulations for non-influenza respiratory viruses of non-human origin will be dictated
through biological risk assessment during the IBC review process.

B7 EMERGENCY PROCEDURES

B7.1

B7.2

B7.3

Illness or Injury Involving Biological Materials
B7.3.1
Severe Injuries
Call 911 for assistance and transportation to the nearest emergency room. Accompany the injured
person to the medical facility and provide information to medical personnel about the
accident/exposure. Report the accident to the PI and EHS.
An incident report will need to be filed immediately so that the individual is eligible for workers
compensation. Access to the online form for Risk Management is available on the EHS website at:
http://www.depts.ttu.edu/ehs/about/incident-reporting.php.Minor incidents not requiring medical
attention should be reported to the PI and then to EHS by using the SCAN system at the following
link: http://www.depts.ttu.edu/ehs/about/scan.php.
B7.3.2
Contamination to the Body
Immediately remove contaminated clothing and flush skin with water. Wash with soap (work into
lather for 30 seconds) and water and flush the area for 5 minutes. Contact the most convenient
local emergency room to obtain care if needed. Report the injury to the PI and to EHS and seek
additional medical assistance if necessary.
An incident report will need to be filed immediately so that the individual is eligible for workers
compensation. Access to the online form for Risk Management is available on the EHS website at:
http://www.depts.ttu.edu/ehs/about/incident-reporting.php.
B7.3.3
Splash to the Eye
Use the emergency eyewash to immediately flush the eye with a gentle stream of clean, temperate
water for 3-5 minutes. Hold the eyelid open. Be careful not to wash the contaminant into the other
eye if it was unaffected by the incident. Contact the most convenient local emergency room to
obtain care if needed. Report the accident to the PI and EHS and seek additional medical
assistance if necessary. NOTE: If chemicals were involved, refer to SDS.
An incident report will need to be filed immediately so that the individual is eligible for workers
compensation. Access to the online form for Risk Management is available on the EHS website at:
http://www.depts.ttu.edu/ehs/about/incident-reporting.php.

B7.4

Fires
1. Without placing yourself in danger, secure biological materials by closing the BSC
sash or placing them in a closed incubator, fridge, or freezer and proceed with the R-A-C-E
process
Remove persons from the immediate area.
Alert others of the situation.
Contain fire and smoke (i.e., shut doors).
Evacuate or extinguish.
2. You are not required to use a fire extinguisher. Only use an extinguisher if you:
(a) Feel confident and not threatened,
(b) Are knowledgeable and TRAINED on how to properly operate a fire extinguisher,
and
(c) Using the fire extinguisher does not put you in danger.
3. Activate the building fire alarm and leave the building at once according to building
evacuation procedures.
4. Meet the fire department outside and tell them of the fire location and details of any
materials potentially involved.
5. Submit a SCAN or Incident Report for any fire:
http://www.depts.ttu.edu/ehs/about/scan.php

B8 DECONTAMINATION AND DISPOSAL

 

B8.1

Factors Contributing to the Destruction/Inactivation of Biological Materials
B8.1.1
Biological factors
1. Microbial load (number and variety of organisms present)
2. Organism life cycle (spore former?)
3. Innate resistance to chemical agent or conditions
4. Presence of biofilms
B8.1.2
Physical and chemical factors
1. Temperature
2. pH
3. Presence of organic and/or inorganic matter.
• Organic matter can create physical barriers reducing contact and bind antimicrobial
agents decreasing overall concentration of the effective agent in a solution or cause
proteins to agglutinate and protect infectious materials. Inorganic matter can create a
physical barrier in the formation of salt crystals.
4. Disinfectant concentration and condition (i.e., expired, mixed with tap water, etc.)
5. Duration of exposure to disinfectant or sterilization process (i.e., contact time).
B8.1.3
Disinfectants must be appropriate for the agent(s) and work.
B8.1.4
Disinfectants must be prepared and used according to the manufacturer’s instructions;
prepared solutions are marked with the expiration date related to the shelf life of prepared
disinfectant per the label.
B8.1.4.1
Bleach solutions are prepared on the day of use unless dictated otherwise in an IBC
protocol.

B8.2

Properties of Chemical Disinfectants
There is no such thing as a perfect chemical disinfectant; there are pros and cons to each.
Common disinfectant types are discussed below and in Appendix BB. Please note that this is not
an exhaustive list of disinfectants.
B8.2.1
Iodophors
Use:
• Recommended dilution is 75-5,000 mg/L (ppm), or approximately 0.5% concentration
• Effective against vegetative bacteria, fungi, and viruses
• Generally safe for stainless steel items and centrifuges
• Some are antiseptics (e.g., betaydyne, scrubodyne)
• Some are surface disinfectants (e.g., Wescodyne™ Steris Corporation)
B8.2.1.1
Pros:
(a) Effectiveness reduced by organic matter (but not as much as with hypochlorites).
(b) Stable in storage if kept cool and tightly covered. Built-in color indicator; if solution is brown
to dark yellow, it is still active.
(c) Generally non-staining
(d) Active in hard water
(e) Relatively harmless to humans
(f) Not as corrosive as chlorine products; leaves a film of residue which allows for residual
antimicrobial activity making it ideal for biosafety cabinets
B8.2.1.2
Cons:
(a) Can stain and discolor equipment in some conditions
(b) Can be corrosive to silver, copper, and aluminum but relatively harmless to stainless steel
(c) Can foam
(d) Cannot be used above 110°F (iodine vaporizes) and is not as effective in low temperature
environments (cold rooms, refrigerators, etc.)
(e) Cannot be used in conjunction with other products
(f) Expensive
B8.2.2
Sodium Hypochlorite (household bleach)
Use:
• User dilution is 1:5 to 1:100 in water; 20% to 1% dilution. Contact time varies with agent to be
neutralized and concentration of solution. Strips to test ppm free chlorine can be purchased.
• Only Clorox brand bleach has been approved by the EPA as a disinfectant; however, any brand
of sodium hypochlorite will meet the requirements of Texas law.
• For chemical disinfection of biowaste a minimum of 1:10 dilution of bleach for no less than 3
minutes is required by Texas law.
• Effective against vegetative bacteria, fungi, most viruses at 1:100 dilution. Contact time varies
with agent to be neutralized and concentration of solution.
• Minimum 1:10 dilution is required for BSL2 activities; 1:5 dilution is needed to inactivate
Mycobacterium and should be used for human materials.
• Available free chlorine is maximized when the solution is pH 5-7.
• Store prepared solutions in brown plastic bottles to protect from light to extend shelf life of
prepared solutions.
B8.2.2.1
Recipe for large quantity ~ 1 % solution with 800 ppm available chlorine:
(a) 1:64 dilution of Clorox in water (2oz in 1 gallon of water)
(b) 2oz of 5% distilled white vinegar (cooking vinegar)
(c) Make daily! (Microbe 6:257, June 2006)
B8.2.2.2
Pros:
(a) Broad spectrum effectiveness
(b) Readily available and inexpensive
(c) High concentrations can kill spores and remove biofilms
B8.2.2.3
Cons:
(a) Contact times and concentration vary with application and agent
(b) Very corrosive, especially to stainless steel
(c) Must be prepared daily for guaranteed ppm of free chlorine
(d) Rapidly inactivated by organic matter, light, and some metals
(e) WARNING!!!! Bleach in combination with other cleaners can produce deadly, toxic
compounds
i. Bleach + 4% phosphoric acid cleaner = chlorine gas
ii. Bleach + Ammonia containing cleaner = chloramine vapors and potentially
hydrazine
B8.2.3
Alcohols (ethanol, isopropanol)
Use:
• The effective dilution for decontamination is 60-80%; 70% is ideal
• Effective against a broad spectrum of bacteria and many viruses
• Ethanol is preferred to isopropanol given it has a slightly more broad-spectrum kill. Ethanol
inactivates all lipophilic viruses and many hydrophilic viruses. Isopropanol is not active against
hydrophilic viruses but virucidal against lipophilic viruses.
• Alcohol waste from submersion must be disposed of as chemical waste
• Use the C1* V1 = C2 * V2 formula to calculate the concentration you wish to make
B8.2.3.1
Pros:
(a) Fast acting and quick drying
(b) Leaves no residue
(c) Relatively inexpensive
(d) Broad spectrum effectiveness against bacteria and viruses
(e) Maintains activity in presence of organic matter
(f) Non-corrosive
B8.2.3.2
Cons:
(a) Not effective against bacterial spores, C. difficile, and Helicobacter
(b) Evaporate rapidly not allowing for extended contact time unless an item is immersed
(c) FLAMMABLE; use only on small surface areas and in well-ventilated areas
(d) Certain agents require a lengthy contact time (30 minutes or more)
(e) Weakens acrylic, polypropylene, PVC and polycarbonate plastics and rubber overtime or
with prolonged or repeated use
(f) Coagulates proteins and can attach them to surfaces (i.e., cannot penetrate protein-rich
materials (e.g., dried blood/plasma)
(g) Can compromise latex and vinyl gloves with extended exposure (1 hour)
(h) Dissolves adhesives in instruments (e.g., microscopes)
(i) Easily passes through nitrile and can carry other chemicals with it.
B8.2.4
Quaternary ammonium salts/Amines (“Quats”)
B8.2.4.1
Use:
• Dilute according to manufacturer instructions
• Spectrum of effectiveness varies with manufacturer; generally effective against Gram positive
bacteria, Gram negative bacteria and enveloped viruses
• Quats sold as hospital-grade disinfectants are generally bactericidal, fungicidal, and virucidal
• Examples include Lysol, BacDown
• 4th generation QUATs maintain effectiveness in the presence of organic material and hard
water
B8.2.4.2
Pros:
(a) Can be used to both clean and sanitize
(b) Non-corrosive
(c) Readily available, generally inexpensive
(d) Low-level human toxicity
(e) Excellent for walls, furniture, and floors
B8.2.4.3
Cons:
(a) Non-sporicidal
(b) Some not effective against non-enveloped viruses, spores or fungi
(c) Hard water and organic matter can reduce effectiveness
(d) Cellulose-containing materials can absorb active ingredients
(e) Some people are prone to allergies and skin-reactivity
(f) Does leave residue
B8.2.5
Phenolics
B8.2.5.1
Use:
• Dilute according to manufacturer instructions
• Effective against bacteria, fungi, and enveloped viruses
• May be a good choice for decontaminating centrifuges which spin blood products
B8.2.5.2
Pros:
(a) Tuberculocidal
(b) Maintain good activity in the presence of organic material and hard water
(c) Residue has some residual activity after drying (follow with EtOH for sensitive applications)
B8.2.5.3
Cons:
(a) Ineffective against non-enveloped viruses, spores and some Gram-negative bacteria
(b) Toxic to infants and the environment
(c) Prolonged exposure can cause allergies and skin irritation
B8.2.6
Hydrogen Peroxide (3-8%) or Vapor-phase Hydrogen Peroxide (VHP)
B8.2.6.1
Use:
• Surface sterilant
• Broad-spectrum effectiveness
• Requires specialized equipment (VHP)
• Aqueous H2O2 concentration 3-8% for spray application and >30% for vaporization
• Vaporization equipment should only be operated by trained personnel. All personnel with
access to equipment or laboratory space should understand the hazards associated with VHP
and signs of exposure. Following SOPs is critical.
B8.2.6.2
Pros:
(a) Environmentally safe by-products (H2O, O2)
(b) Rapid kill action
(c) No disposal issues, odor or irritation when diluted (3-8%)
(d) Readily available and inexpensive
(e) Good compatibility with sensitive equipment, electronics and furnishings
(f) VHP offers low temperature sterilization and is a safer alternative to formaldehyde or
ethylene oxide gas
(g) VHP is cost effective after initial setup
B8.2.6.3
Cons:
(a) Little penetration
(b) Concentrations >7.5% can cause discoloration of metal finishes
(c) Compatibility concerns with brass, zinc, copper, and nickel/silver plating
(d) Oxidizing capability is rapidly inactivated by organic material; cellulose cannot be processed
(e) Nylon items can become brittle
(f) Vapors have no color and are odorless; inadvertent exposure can cause serious health
effects
(g) VHP is expensive to implement

B8.3

Management of Biologically Contaminated Materials and Biowaste
The treatment and disposal of biological waste (i.e., biowaste) is regulated by TCEQ.
B8.3.1
Work areas which generate biological waste must segregate biological waste from other
hazardous wastes and general, non-hazardous (municipal) wastes for treatment.
B8.3.1.1
Biologically contaminated materials and biowastes are not transported in public areas
without secondary containment (see B8.6.3.1) or left uncontained or unattended prior to
initiation of disinfection.
B8.3.2
Collection and/or Disposal Containers
Each PI which produces biowaste is responsible for purchasing appropriate containers in
an adequate number and biohazard bags for the collection, containment, and disposal of
biological wastes generated.
B8.3.2.1
The number of containers should be such that a container is easily accessible from the
immediate workstation so that containers are not moved about the space to accommodate
waste.
B8.3.2.1(a)
Sharps Containers
Use FDA-approved sharps containers to dispose of sharps & contaminated glass in
biological laboratories. They are available in various sizes and have the following features:
i. Puncture-resistant rigid plastic
ii. Red
iii. Labeled as containing a “Biohazard” and "Sharps"
iv. Lid design traps items so they cannot be retrieved, is puncture/leak resistant and locks
tightly
v. Have a line that indicates when the container is full.
These containers may be purchased from local sources, including the Physical Plant Central
Warehouse, Chemistry Stockroom, and medical supply stores, as well as from laboratory product
distributors.
B8.3.2.1(b)
Do not purchase or use "needle-cutter" devices; use of such devices can generate aerosols
and leaves sharp barbs.
B8.3.2.2
Biohazard / Autoclave Bags and Holders
Autoclave bags (commonly referred to as “biobags or biohazard bags”) can be purchased from
laboratory product distributors in a variety of sizes and thicknesses. These bags are tear resistant
but can be punctured; they may also burst in the autoclave when sealed because they are
impermeable to steam.
B8.3.2.2(a)
Biobags must be made of polypropylene.
B8.3.2.2(b)
Biobags must have a biohazard symbol; the bag can be purchased in red, orange, and clear.
Avoid yellow biohazard bags; yellow is associated with other types of hazardous waste on campus.
B8.3.2.3
Except for bench-top pipette tip collection bags, biobags must be placed inside a rigid
container with closable lid (latching or step can style) labeled with the biohazard symbol
while waste is being collected.
B8.3.2.3(a)
If a normal trash bin is used to hold a biohazard bag, the trash bin must: 1) Be labeled with
a biohazard symbol, 2) Never used for non-biohazardous waste, 3) and have inner and outer
surfaces that are able to be disinfected.
B8.3.2.4
Benchtop biowaste collection must be managed in one of the following ways:
(a) wire bag holders with or without the optional lid;
(b) labeled beaker with appropriately diluted bleach or other EPA-approved disinfectant
for the agent(s) that is disposed of at the end of work; and/or
(c) beaker or other container that is lined with a biohazard bag. If the container does not
allow view of the biohazard symbol on the bag it must be labeled with a biohazard
sticker.
B8.3.2.4(d)
Benchtop biowaste collection must be done using materials that withstand frequent surface
disinfection. Biokeeper boxes and similar containers are one-time use and must be closed
and disposed of as biowaste once full rather than emptied and reused.
B8.3.2.5
Biowaste must not exceed three-quarters of the volume of the bag and/or container.

B8.4

Treatment of Biologically Contaminated Materials and Biowaste
As stated in B8.3.1, all biological wastes must be segregated from other waste streams and require
treatment prior to disposal.
Thus, to facilitate regulatory compliance and consistency of treatment practices, EHS has instituted
a waste program that includes autoclave use and monitoring (see section B8.6.4).
Regulations Pertaining to the Treatment of Biowaste
The following is taken directly from the Texas Administration Code (TAC); please refer to 30 TAC
§326, 30 TAC §330 and 25 TAC §1.132 for definitions related to the regulations. For example,
“bulk” according to the TAC consists of amounts greater than or equal to 100 mL.
Medical waste consists of waste associated with many biological laboratory activities and includes:
animal waste, bulk blood, bulk human blood, bulk human body fluids, microbiological waste,
pathological waste, and materials listed in 49 Code of Federal Regulations, Part 173, §173.134(a).
The term does not include medical waste produced on a farm or ranch as defined in 34 TAC
§3.296(f) (relating to Agriculture, Animal Life, Feed, Seed, Plants, and Fertilizer).
B8.4.1
According to the TAC, “medical waste” (i.e., biowaste) requires treatment prior to final
disposal. This treatment may be performed by the generator or transferred offsite and
performed by a third party.
B8.4.2
Biowaste to be transferred to a third party for treatment must be properly prepared for
transport by the generator (i.e., proper preparation of biobarrels). See Appendix BG.
B8.4.3
Biowaste that is treated by the generator must be treated using a TCEQ-approved method
of treatment for the specific type of biowaste. Approved methods are listed in 25 TAC §1.136.
Methods accessible at TTU include chemical disinfection with an approved disinfectant or steam
disinfection (i.e., autoclaving).
B8.4.4
A written SOP for biowaste treatment, including preparation of any chemicals used and
preparation of materials for autoclaving, must be in place in work areas treating their
biowaste.
B8.4.5
Treatment of all biowaste must be recorded. The biowaste treatment record must be
maintained by the generator for no less than 3 years.
Biowaste treatment log pages that meet the state requirements are available in in Appendix BF or
can be downloaded from the EHS website: http://www.depts.ttu.edu/ehs/academicsafety/lab/toolstemplates.php
B8.4.5.1
Record of Biowaste Treatment
Laboratories must maintain a written record that, at a minimum, contains the following
information for each batch of waste treated:
(a) Date of treatment;
(b) Amount of biowaste treated;
(c) Method/conditions of treatment;
(d) The name (printed) and initials of the person(s) performing treatment; and
(e) The written procedure for the operation and testing of any equipment used and a written
procedure for the preparation of any chemicals used in treatment.

B8.5

Regulations pertaining to chemical disinfection of biological wastes
Failure to properly follow TCEQ biowaste regulations can result in fines to the University.
B8.5.1
Only EPA-registered disinfectants approved by the Texas Department of Agriculture or
properly diluted household bleach (1:10 household bleach to water minimum) shall be used
for chemical decontamination of biological wastes.
B8.5.1.1
Freshly prepared disinfectants are required for chemical treatment of biowastes.
B8.5.1.2
Expired stock or dilutions of disinfectants shall not be used for any purpose.
B8.5.2
Should you choose to decontaminate liquid biowaste by chemical disinfection, a validated
standard operating procedure for the specific liquid matrix to be decontaminated must be
followed.
B8.5.2.1
The method must be validated by the PI according to 25 TAC §1.135.
B8.5.3
All label instructions for dilution and contact time must be followed.
In failing to do so, the PI assumes full liability for any incidents/injuries which result from off-label
use and is potentially subject to enforcement action under the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA).
B8.5.4
All stock solutions of chemical disinfectants shall be stored according to the label/SDS. Do
not store solutions of incompatible chemicals under sinks (e.g., bleach with ammonia-containing
cleaners). Consult the label for disposal. If you have questions, call EHS.
B8.5.5
When diluted household bleach is used for chemical disinfection of biowaste, a 1:10
dilution of bleach to water is freshly prepared (B8.5.1) and items shall be totally immersed
for a period of time not less than three minutes or as required by the work-specific protocol
– whichever is longer.
B8.5.6
Solid biowaste immersed in a liquid chemical agent must be thoroughly drained before
disposal.
B8.5.6.1
Agars and related biological substrates are not considered solid wastes capable of
chemical disinfection.

B8.6 Steam Disinfection (Autoclaving)

Autoclaves use pressurized steam to destroy microorganisms and are the most widely used and
accepted system available for the decontamination of laboratory biowaste and the sterilization of
laboratory equipment, glassware, media, and reagents.
There are four parameters which must be met for effective steam sterilization: steam, pressure,
temperature, and time. Should a cycle fall short in reaching the minimum target required in one of
these areas, sterilization will not be accomplished.
To ensure consistent sterilization practices, EHS has instituted a program for autoclave use and
monitoring. Mechanical, chemical, and biological monitors should be used to evaluate unit
performance in accordance with the program and TCEQ regulations.
B8.6.1
Autoclaving should be used, whenever possible, as the preferred means of
decontamination of laboratory biowaste. See B8.6.7.2 and B8.6.7.3 for proper autoclaving of
bagged biowaste.
B8.6.2
It is the PI’s responsibility to ensure operator competence and devise SOPs for the proper
methods of cleaning instruments, preparing packages, loading and operating the autoclave,
and maintaining an autoclave log that meets the requirements of B8.4.5.1.
B8.6.2.1
Those using building autoclaves are to record biowaste treatment runs in the provided
logbooks.
B8.6.3
Equipment Specifications
Pressure-cooker style autoclaves and home-built reactor pressure vessels are not to be
used.
B8.6.3.1
Autoclave Pressure Relief Devices
All autoclaves and reactor pressure vessels must be protected with a rupture disc as the
primary pressure relief device.
Rupture discs are thin metal discs which are designed to burst at a specific pressure. Spring
loaded relief valves may be used as a secondary pressure relief device.
B8.6.3.2
Precautionary notes regarding autoclaves
Many new laboratory pressure reaction vessels will not be stamped with the ASME or CE
marking. Choosing manufacturers that are known to follow ASME codes is a way to
ensure that pressure vessels meet manufacturing requirements.
Modifications to pressure vessels may impede or impair designed safety features.
B8.6.4
Regulations Pertaining to Steam Disinfection of Biological Wastes (autoclaving)
B8.6.4.1
Biowaste that must be transported outside of the laboratory for autoclaving must be
transported in a durable, leak-proof container labeled with the biowaste symbol.
B8.6.4.2
To allow sufficient steam access to and penetration of the biowaste, the biowaste shall be
packaged according to the recommendations provided by the manufacturer and loaded into
the chamber so as to not exceed the capacity limits as set by the manufacturer.
B8.6.4.2(a)
Bags to be autoclaved must be secured at the top such that steam is able to enter and vent
from the bag.
Do not twist, knot, or tape the bag shut in such a way that prevents steam entering/escaping the
bag.
B8.6.4.2(b)
Chemical indicators (e.g., autoclave tape) must be used in association with autoclaved
biowaste.
B8.6.4.3
For biowaste treatment, the temperature in the chamber of the autoclave must reach at least
121°C and there must be at least 15 pounds per square inch gauge pressure for at least 30
minutes. The 30 minutes does not include unit pressurization and depressurization, only time held
at 121°C (25 TAC §1.133).
B8.6.4.4
The autoclave must be operated according to the manufacturer’s instructions.
B8.6.4.5
Any markings that identify the waste as a biowaste (e.g., biohazard symbol) shall be
covered with a label that identifies the waste as treated medical waste.
B8.6.4.5(a)
A label that states the contents of the disposable container have been treated in
accordance with the provisions of 25 TAC §1.136 must be affixed to the biowaste bag over
the biowaste symbol. Contact EHS for labels.
B8.6.4.5(b)
The treated and labeled biowaste bag must be placed in a black garbage bag and disposed
of in the municipal waste dumpsters by laboratory personnel.
B8.6.4.5(c)
Treated biohazard bags must be secured in a black bag before final deposition in the
building dumpster.
B8.6.4.6
Required Frequency of Biological Testing
B8.6.4.6(a)
For building-supported autoclaves, the department representative or building manager in
conjunction with EHS shall:
i. Demonstrate a minimum four log ten reduction (10^4 log) (as defined in 25 TAC §1.135
(relating to Definitions)) on routine performance testing using a biological indicator
containing pertinent Bacillus species (as defined in 25 TAC §1.135).
ii. Conduct testing on autoclaves which process the quantities listed below at the applicable
intervals:
• For generators of more than 50 pounds but less than or equal to 100 pounds per month,
testing shall be conducted at least monthly;
• For generators of more than 100 pounds but less than or equal to 200 pounds per month,
testing shall be conducted at least biweekly; and
• For generators of more than 200 pounds per month and persons that treat medical wastes
off-site, testing shall be conducted at least weekly.
B8.6.4.6(b)
EHS will facilitate annual testing of PI-owned autoclaves. PIs who have autoclaves in their
laboratories which process biologically contaminated materials and/or biowaste is
responsible for conducting testing at the applicable frequency listed in B8.6.4.6(a)ii as it
applies to their waste throughput. Contact EHS for biological indicators.
B8.6.5
Monitoring Autoclave Performance
As indicated above, autoclaves used to decontaminate laboratory biowaste must be tested
periodically to assure effectiveness to protect human health and the environment.
B8.6.5.1
Mechanical monitors include thermometers, pressure gauges and displays on the autoclave.
Correct readings do not ensure sterilization; but incorrect readings could be an indication of
problems.
EHS recommends utilizing the printer for autoclaves as a regular means to monitor and log
autoclave performance. A cycle print-out is available on most campus units; printers on
these units should be maintained with ink and paper. EHS strongly recommends printers on
campus units.
B8.6.5.2
Autoclave performance shall be evaluated with chemical and biological tests. Indicators
shall be placed in autoclave locations that are the slowest to heat.
There are two types of tests are frequently used to evaluate autoclave efficacy:
(a) Chemical indicators
(b) Biological indicators
Please see the Appendix BE for forms and the SOP for the Autoclave Testing Program. If you
need assistance or have questions about monitoring, please contact EHS at 806-742-3876.
B8.6.5.2(a)
Chemical Indicators
As stated in B8.6.4.2 (b), heat-sensitive indicator tape or an equivalent chemical indicator shall be
used when autoclaving biowaste materials.
Chemical indicators use temperature-, pressure- and/or vapor- sensitive chemicals to assess
physical conditions during autoclaving and are generally inexpensive and easy to use. Multiparameter and single-parameter chemical indicators are available for autoclaves. Refer to the
manufacture for placement of chemical indicators within the load. Heat-sensitive indicator tape is a
chemical indicator and should be used on all materials within every autoclave load.
Chemical indicators should be used in conjunction with biological indicators - not replace them.
Chemical indicators only assess if the physical parameters of sterilization were met; biological
indicators are the only means to prove sterilization has been achieved.
B8.6.5.2(b)
Biological Indicators
Biological indicators contain heat-resistant spores that are destroyed when an autoclave is
functioning properly (generally Geobacillus stearothermophilus for heat treatment. Other spore
types are used for chemical sterilization.). Biological indicators are the most accepted and widely
used means of monitoring sterilization processes.
EHS provides biological indicators for state-required testing and assessing autoclaves after
repairs. EHS will test all University autoclaves with biological indicators at least annually.
Autoclave testing frequency will follow state guidelines at minimum (See B8.6.4.6(a) ii). The
procedure and treatment log sheets can be found in Appendices BE and BF.
Biological indicators shall be used when:
i. The autoclave has been repaired
ii. New waste cycle parameters (i.e., settings) are set
iii. Biowaste loading procedures or materials have changed
Biological indicators should also be used when:
i. A new type of packaging material or tray is used
ii. New personnel are trained
NOTE: These evaluations fall outside the periodic testing facilitated by EHS and will be at the
laboratory’s expense.
B8.6.5.2(c)
Failed Indicators
If an autoclave fails a biological test:
i. Post an “out of service” sign on the unit and contact the appropriate party to initiate a
service call.
ii. Do not use the autoclave until it has been inspected, repaired, and successfully challenged
with a biological indicator in 1-3 consecutive “dummy” or non-hazardous loads with control
tests for each.
iii. Off-campus and PI-owned units will need to record and report all results to EHS. Please
see the procedure in Appendix BE and contact EHS with further questions.
B8.6.6
Autoclave Container Selection
B8.6.6.1
Polypropylene Containers and Pans
Polypropylene (recycle #5) is a plastic capable of withstanding autoclaving but resistant to heat
transfer. Therefore, materials contained in a polypropylene pan will take longer to autoclave than
the same materials in a stainless-steel pan.
Do not use polyethylene (recycle #1) or high-density polyethylene (recycle #2) plastics for
autoclaving.
B8.6.6.2
Stainless steel Containers, Pans, and Baskets
Stainless steel is a good conductor of heat and is less likely to increase sterilizing time; however, it
is more expensive than polypropylene.
B8.6.7
Preparation and Loading of Materials
For efficient heat transfer, steam must flush the air out of the autoclave chamber.
Before using the autoclave, check the drain screen at the bottom of the chamber and clean
it if it is blocked. If the sieve is blocked with debris, a layer of air may form at the bottom of the
autoclave, preventing efficient operation.
B8.6.7.1
Liquids
(a) Fill liquid containers only half full. Loosen caps or use vented closures. Avoid large bottles
with narrow necks and sealed containers as they may explode.
(b) Cover bottles without a lid loosely with aluminum foil.
(c) Place bottles in an autoclavable secondary container to catch spills. Add water to the
bottom of the container to reduce the likelihood of cold spots in processing. See section
B8.6.6.
B8.6.7.2
Packets and Bags
(a) Always put bags of biological waste into an autoclavable secondary container to catch
spills. See section B8.6.6 for guidance regarding secondary containers.
(b) Fill bags no more than three-quarters full. Do not over fill bags.
(c) Position biohazard bags on their sides, with the bag neck closed loosely.
i. Use indicator tape or twist ties to loosely secure the top of the bag.
ii. Do not overfill bags or twist/knot the tops of bags. This prevents adequate steam
entry into the bag and may result in incomplete sterilization of materials.
(d) In the case of a vial, insert a blunted needle into the septum.
(e) Place a strip of indicator tape on bag if not otherwise used to secure the top of the bag.
(f) Add water to the bottom of the bin to facilitate heat
(g) Packaging materials for packets must allow steam penetration and maintain sterility after
processing. There are several methods to accomplish this. Call EHS at 806-742-3876 if
you have questions.
B8.6.7.3
Autoclave Loading
B8.6.7.3(a)
Loading procedures must allow adequate steam circulation and will vary with items to be
autoclaved and unit configuration. Do not exceed the capacity of the unit. (25 TAC §1.133).
Refer to your unit’s user guide if you have questions regarding either of these procedures.
B8.6.7.3(b)
Wear lab coat, eye protection, heat-protective gloves, and solid shoes. Carry your lab coat
on a cart- do not wear your PPE if you must travel in public corridors to the autoclave.
i. Select containers with the lowest sides and widest diameter possible for the autoclave.
ii. Remove container lid if present.
iii. Leave space around each item.
iv. Drill a hole inside of container or pour water in the bottom of the tub to facilitate heat
transfer, add moisture to dry loads, and eliminate the air gap.
v. Use perforated trays and/or wire baskets for items that will not generate spills.
B8.6.8
Cycle Selection Guidance
• Use liquid cycle (slow exhaust) when autoclaving liquids, to prevent contents from boiling over.
• Select fast exhaust cycle for glassware.
• Use fast exhaust and dry cycle for wrapped items.
B8.6.9
Time Selection Guidance
(a) Consider the size of the articles to be autoclaved and the overall amount in the load. For
example, a 2-liter flask containing 1 liter of liquid takes longer to sterilize than four 500 mL
flasks each containing 250 mL of liquid and 4 flasks containing 1 liter will take less time than
10 flasks containing 1 liter.
(b) Material with a high insulating capacity, such as animal bedding and high-sided
polypropylene containers, increases the time needed for the load to reach sterilizing
temperatures thus the load will require extended time to achieve sterilization.
(c) All biologically contaminated materials should be autoclaved for a minimum of 30 minutes at
121°C and 15 psi. The 30-minute minimum does not include heat and pressurization
priming or depressurization; account for these periods separately when calculating total
cycle time for your unit if it is not already programed.
B8.6.10
Removing the Load
(a) Wear lab coat, eye protection, heat-protective gloves, and solid shoes. Carry your lab coat
on the cart - do not wear your PPE if you must travel in public corridors to the autoclave.
(b) Check that the chamber pressure ias zero.
(c) Standing behind the door while you open it, slowly crack open door to vent steam. Beware
of this rush of steam.
(d) If liquids were processed, open autoclave door and allow liquids to cool for 20 minutes
before removing.

B8.7

Summary of Biological Waste Treatment and Disposal by Generators
By law, biowaste is to be treated in accordance with 25 TAC 1.136 (B8.4.3). Please contact EHS if
you have questions as to how to address your biologically contaminated materials or biowaste.
Steam Disinfection of Solids
• Collect disposable, solid materials contaminated by a biological agent, excluding sharps, into
an autoclave bag within an appropriate container.
• When half to three-quarters full, prepare bag as described in B8.6.7.2 and autoclave the bag as
described in section B8.6.4.3.
• Allow bag to cool, affix a “treated” sticker to the bag and place autoclave bags in black trash
bags. Contact EHS if you need more “treated” stickers (See B8.6.3.5(a)).
• Laboratory staff dispose of treated waste in the building's outdoor dumpster.
Chemical Disinfection of Solids
This method is for reusable laboratory supplies, pipet tips, serological pipets, reusable syringes
and items of that nature – not petri dishes with cultures, contaminated food products and similar
biowastes. Contact EHS for guidance and if you have any questions about treating your biowaste.
• A pre-cleaning step to remove debris may be required for certain items.
• Prepare qualifying disinfectant per the manufacturer’s guidelines or a 1:10 dilution of household
bleach and water.
• Fully submerge biologically contaminated materials in solution (i.e., suck solution into pipet
tips). All surfaces must have contact with the disinfectant.
• Allow contact time per the manufacturer’s instructions, or if bleach is used, no less than 3
minutes or according to the lab-validated waste disinfection method - whichever is longer.
Different agents require different contact times with bleach. Contact EHS if you plan on
chemically disinfecting your biologically contaminated materials or biowaste.
Steam Disinfection of Liquids
If liquid biowaste is mixed waste, contact EHS. Autoclaving and chemical disinfection may not be
recommended. Contact EHS for the final disposal of your treated liquid waste. Some items are
not accepted in the sanitary sewer or can damage plumbing.
• Fill liquid containers only half full. Loosen caps or use vented closures. Avoid large bottles
with narrow necks when possible.
• Cover bottles without a lid loosely with aluminum foil.
• Place bottles in an autoclavable secondary container to catch spills.
• Autoclave for at least 30 min at 121°C and 15 psi of pressure on a cycle with slow exhaust.
The 30min time point does not include warm up or cool down.
• Allow liquids to cool before final disposal.
Chemical Disinfection of Liquids
Contact EHS if you plan to not use a bleach solution to disinfect your biowaste. Make sure that
bleach will not adversely react (e.g., produce chlorine gas) with your biowaste.
Should you choose to decontaminate liquids by chemical disinfection, a validated standard
operating procedure for the specific liquid matrix must be followed. Different materials/agents
require different bleach concentrations and contact times.
• Add, at minimum, an equivalent volume of freshly prepared 10-20% household bleach solution
to the container with the liquid waste. Gently mix.
• Allow contact time per SOP.
• Dispose of solution by pouring down the sink.
• Flush with copious amounts of water to prevent damage to plumbing.
• Wash container as usual and rinse well before further use.

B8.8

Biological Wastes Picked up by EHS
B8.8.1
Sharps Waste
Sharps, according to 25 TAC §1.132 include but are not limited to the following when
contaminated: hypodermic needles; hypodermic syringes with attached needles; scalpel blades,
razor blades, disposable razors, and disposable scissors used in surgery, labor and delivery, or
other medical procedures; intravenous stylets and rigid introducers (e.g., J wires); glass Pasteur
pipettes, glass pipettes, specimen tubes, blood culture bottles, and microscope slides; broken
glass from laboratories; and tattoo needles, acupuncture needles, and electrolysis needles.
Regardless of contamination, hypodermic needles and hypodermic syringes with attached needles
are considered sharps.
Sharps must be segregated from other wastes.
B8.8.1.1
Safe Disposal of Sharps:
(a) FDA-approved containers (i.e., puncture-proof and securable) are required for the disposal
of sharps.
(b) Use care and caution when cleaning up after procedures that require the use of sharps.
(c) Do not recap or remove needles from syringes. Recapping requires an EHS-approved
SOP.
(d) Do not recap or remove scalpel blades by hand.
(e) Discard needle and syringe as an intact unit immediately after use into puncture-resistant
sharps containers.
(f) Do not overfill sharps containers. Replace when waste reaches indicator line on container
or when 3/4 full.
(g) Dispose of ALL sharps containers through EHS waste disposal. “Biological” is the indicated
waste type unless radioactive materials are present.
i. Sharps that have contacted radioactive materials require a separate sharps
container appropriately labeled for the radioactive hazard. When full, dispose of
sharps container through EHS as “Radioactive” waste. See the Radiation Safety
Manual for details.
In the event of a sharps-associated injury, notify the PI and/or senior lab personnel and seek
treatment if needed. Fill out and turn in an incident report within 24hrs of the injury. Access to the
online form for Risk Management is available on the EHS website at:
http://www.depts.ttu.edu/ehs/about/incident-reporting.php.
Call EHS if you have any questions at 806-742-3876.
B8.8.2
Biological Waste Barrels (biobarrel)
Biological waste barrels with liners are provided by EHS to laboratories for biological waste
disposal in certain cases. Laboratories that may need barrels might include those which:
1. Lack access to an autoclave or the autoclave is out of service;
2. Perform research which periodically generates large amounts of biowaste that is not
manageable in available autoclave(s);
3. Perform research which includes inoculated or naturally infected live animals, whole
carcasses of unpreserved animals, whole cuts of meat or other dense items which will not
autoclave properly.
4. Other special cases as determine by EHS (mixed waste, high hazard, etc.).
B8.8.2.1
Biobarrel Use
Biobarrel use is addressed in EHS SOP 6.5, Use of EHS-provided Biobarrels. Violations of the
points below can result in delayed waste pick up or revocation of barrel privileges in locations of
elected barrel use. See Appendix BG for details.
(a) Properly segregate biowaste from non-biowaste.
(b) Accumulate biowaste in smaller, appropriate biowaste receptacles. Do not place
loose materials directly into the biobarrel.
(c) Do not place large quantities of liquid biowaste into biobarrels (e.g., spent cell-culture
media, liquid microbial culture media, supernatants, etc.).
(d) Secure individual biowaste bags prior to placement in the biobarrel.
(e) Tie off biobarrel bag liner when biobarrel is ¾ full. Biobarrels cannot weigh more
than 40 lbs.
(f) Avoid compressing waste in the biobarrel.
(g) Biobarrels are to remain closed except when a biowaste bag is actively being
transferred to biobarrel.
B8.8.2.2
Biobarrel Pick-Up
(a) Liners must be “tied off” (i.e., knotted – sides pulled up and tied together)
(b) Lid must be secured.
(c) Weight must not exceed 40lbs.
(d) Submit biowaste pick-up request online through the EHS website:
http://www.depts.ttu.edu/ehs/.
(e) The number of barrels picked up will be the number of barrels replaced. This number can
be modified in the comment section of the waste request, but the requested number may
not necessarily be granted.
A biobarrel usage poster is available on the EHS website:
https://www.depts.ttu.edu/ehs/academicsafety/lab/tools-templates.php. You can also contact EHS
for a printed copy.
B8.8.3
Multi-hazard or Mixed Waste
Avoid generating mixed waste; if unavoidable, keep volume of mixed waste to a minimum.
Do not steam or chemically disinfect mixed waste without EHS consult.

B8.9

Disposal of Animal Tissues, Carcasses and Bedding
B8.9.1
Disposal of non-farm/agriculture-related animal tissues, unpreserved carcasses and bedding is to
be coordinated with EHS and ACS. Freeze / refrigerate tissues and carcasses until pick-up to
prevent odor as needed.
B8.9.2
Preserved carcasses, drained of preservatives, can be disposed of as municipal waste.
B8.9.2.1
Collect all liquid preservatives and dispose of them as hazardous waste. Place carcasses
in a black trash bag and dispose of it in the building’s dumpster.
If you have questions, please call EHS at 806-742-3876.

B8.10

Plant and Seed Devitalization
Genetically modified plants and seeds must be rendered non-viable prior to disposal. Some
collaborators or funding agencies may require a maceration step or incineration.

B8.11

Special Considerations for Toxin Neutralization
Neutralization methods must be specific to the toxin and will be described in the IBC
protocol.

B8.12

Special Considerations for Prion/Prion-like Protein Neutralization
Neutralization methods must be specific to the prion/prion-like protein and will be described
in detail the IBC protocol.

B8.13

Other Waste Issues in Biological Laboratories
B8.13.1
Reusable Labware
Items such as contaminated culture flasks, media bottles, test tubes, instruments, equipment
tubing, etc. are decontaminated by lab personnel before washing (and repackaging) by one of two
methods:
• Autoclave items that have been collected in autoclavable containers.
• Chemically disinfect items by soaking in a fresh 10% sodium hypochlorite solution for a
minimum of 20 minutes before washing. For heavily soiled items, remove as much of the
visible contamination as possible over an autoclave bag (for autoclaving or EHS waste
collection) then soak in fresh 10% household bleach. Repeat as necessary. Follow
disinfectant step(s) with a thorough water rinse. The rinse is especially important for items
vulnerable to corrosion and remove bleach residue.
B8.13.2
Glass
B8.13.2.1
Non-contaminated glass waste must be segregated from other solid municipal waste and
disposed of in a sturdy, puncture-resistant, closable box labeled as broken glass (CHP,
A19.3.4.1).
There are autoclavable, benchtop boxes you can purchase for easy disposal of pipettes, slides and
other small glass items (Terminal® Biohazard Benchtop Keeper™ and Terminal® Pipet Keeper™
Containers, Whitney Products).
B8.13.2.2
Full glass disposal boxes should be taped closed and disposed of in a dumpster by the
laboratory (i.e., accumulator) (CHP, A19.5.2).
B8.13.2.3
Do not handle broken glass with your hands. Use forceps, tongs, or some other tool to
collect and handle broken glass. Decontaminate collection tools after collection of
contaminated glass.
B8.13.2.4
Biologically contaminated glassware should be immediately disposed of in a sharps
container. This minimizes handling of the contaminated hazard.
If disposal in a sharps container is not possible, glass must be decontaminated before
disposal in the glass disposal box (CHP, A19.5.1). Glass can be decontaminated by steam of
chemical disinfection. Autoclaving is the recommended decontamination method.
(a) Autoclave in a puncture-proof, autoclavable container; or
(b) Place in a wire sieve then soak in freshly prepared 10% household bleach solution or other
approved chemical disinfectant solution for the appropriate contact time.

B9 BIOLOGICAL LABORATORY OPERATION

Biological risk management approaches vary with the agent(s), procedure(s), and location(s)
associated with the work. Adequate and appropriate risk management is essential to the safe
operation of the biological laboratory.

B9.1 Risk Management

Risks must be identified and addressed prior to beginning laboratory work and repeated
when any changes are made to the agent, procedure, equipment, employee, or facility.
All biological work carries some level of risk. Worker training and competency are paramount for
personnel, laboratory, and environmental safety.
Biological risk assessment (RA) is a continuous and evolving process dependent on the quality
and quantity of current information and recommendations of appropriate, realistic methods of
containment. A RA should be performed by those most knowledgeable of the agent or technique
and is thus a pivotal responsibility of PIs and is shared and supported by EHS, the IBC, and other
Institutional Committees as applicable.
Information identified in a RA will provide a framework for appropriate PPE, laboratory practices,
engineering controls and safety equipment to manage the identified risks. The three basic steps to
risk management are hazard identification, risk assessment and risk control.
B9.1.1
Identification of Hazards
1. Identify the agent(s) if they are known. Samples of unknown status and environmental
samples should be handled with universal precautions at BSL2 unless permission has been
granted otherwise by the IBC (see B6.2.2).
2. Identify all procedures and equipment which will be applied to the agent.
3. Identify the location(s) where work will be done.
4. Identify who will be performing the work.
Identification of the above research elements will help you to properly evaluate the three central
components of a RA (agent, environment, and host) which need to be addressed when considering
biological risks.
Several factors about each component and relationships
between components are evaluated in step 2: Risk
Assessment.
B9.1.2
Biological Risk Assessment (RA)
Biological risk assessment is generally not as prescriptive as
a risk assessment for a chemical or physical hazard as it
involves living organisms that exist in a state of natural
variation.
FIGURE1. Risk Assessment Triad
Barbara Johnson, PhD, RBP - Understanding, Assessing, and
Communicating Topics Related to Risk in Biomedical Research
Facilities, ABSA Anthology of Biosafety IV - Issues in Public
Health, Chapter 10 (2001), http://www.absa.org/0100johnson.html.
B9.1.2.1
Biological Agent(s)
Given the ubiquitous nature of some microbes, it is important to consider naturally occurring strain
variation and the potential for differential expression of virulence factors.
This variation, in conjunction with the dynamics of the host-agent relationship, complicates
calculating infectious dose; however, relative estimates can be made regarding the risk an agent
poses when the following elements of an agent are considered:
• Pathogenicity and resilience
• Naturally occurring mode(s) of transmission
• Likely mode(s) of transmission in the laboratory
• Natural reservoirs
• Host range (e.g., zoonosis or reverse zoonosis)
• Vectors
• Availability of treatment/prevention measures
See B6.2 regarding risk groups. Risk groups are a starting point for a RA based on what is known
about an agent; however, risk group alone is not a substitute for a RA.
B9.1.2.1(a)
Additional Factors to Consider Concerning Biological Agents
• Concentration (natural burden vs lab culture)
• Volume
• Exposure associated with procedures (i.e., aerosolization, injection, involvement of vectors or
animals, etc.)
• Life cycle stage(s) to be encountered/manipulated
These factors may increase/decrease the containment level required for an agent regardless of the
risk group (B6.2.1).
B9.1.2.2
The Environment
Environmental factors to be considered in a RA primarily concern agent containment and
environmental impact. Environmental factors may include:
• Ventilation and laboratory design
• Laboratory procedures and training
• Containment equipment
• Established and updated operating procedures
• Personal Protective Equipment availability and usage
• Laboratory sanitation and biowaste management
• Use of animals and/or vectors
• Presence of host species and/or agent vectors outside containment
• Field work
B9.1.2.3
The Host
The human factor is the most difficult hazard to control.
Lab personnel are encouraged to alert their PI or laboratory supervisor if they have a health
condition that can affect their safety in the lab so that duties can be properly assigned.
Lab personnel are strongly encouraged to inform their personal physician of all hazardous
material they handle in the laboratory.
Lab personnel and visitors are to be informed of the hazards to which they may be exposed;
this includes potential routes of exposure, symptoms, and treatment options and availability.
B9.1.2.3(a)
The human immune system can be affected by multiple factors that can modify the host response
to an exposure; such factors may include but are not limited to:
• Genetic predisposition
• Pregnancy
• Stress and fatigue
• Medications
• Chronic conditions (e.g., diabetes, HIV, allergies, autoimmune disease, etc.)
• Acute conditions (e.g., common cold, open wound/laceration)
• Treatment sensitivities
• Vaccination status
• Age
• Composition of natural flora
B9.1.2.3(b)
Some medications may alter your state of mind leaving you less coordinated, alert, and capable in
the laboratory.
B9.1.2.3(c)
You are only as safe as those with whom you work. Behavioral factors that can affect worker
safety in the laboratory include:
• Education
• Lack of training or recurrent training or poor training
• Failure to use PPE, use of PPE in poor condition, or improper use of PPE
• Poor or improper laboratory habits (lab-specific safety culture)
• Complacency
• Distractions
Elimination
Substitution
Engineering
Administrative
Behavior
PPE
B9.1.2.3(d)
The above primarily focuses on humans as a host. Host range (plant species and non-human
animal species) must also be explored as it pertains to the agent(s).
B9.1.2.4
Risk Matrices
While assessing risks, it is also important to prioritize the risks you are evaluating and direct
attention to those with the greatest probability of occurrence and can cause the most harm. The
risk matrices below can help guide prioritization.
The agent risk groups are listed across the top. The hazard level of the procedure to be used is on
the far left; procedures that use aerosol-generating equipment or sharps would be considered to
have a high probability of an accident/incident occurring.
TABLE 2. Example Risk Matrix Regarding Agent
Table adapted from Biological Safety: Principles
and Practices 4th Ed., Fleming and Hunt,
ASM Press 2006.
B9.1.3
Risk Control
Once hazards are identified and the risk they pose assessed,
control measures can be implemented. Hazard controls may
be on an administrative, facility, or laboratory level and can
range from alternate methodologies or PPE to facility design
and biosafety cabinets.
A key element in risk control is the laboratory-specific
biosafety manual, also referred to as a Biological Work Area
Safety Plan (bioWASP/WASP). This document may either be
an amendment to an existing WASP or a stand-alone
biosafety-specific document in addition to your general WASP
or your IBC protocol.
The (bio)WASP serves as the foundation for administrative risk
control measures (Administrative Controls) as it addresses
laboratory and facility operations, training requirements,
FIGURE 2. Hierarchy of Controls
NIOSH, Hierarchy of controls.
https://www.cdc.gov/niosh/topics/hierarchy/default.html
application of primary and secondary containment measures, emergency procedures, exposure and
health monitoring guidelines and any other necessary materials for a safe laboratory environment.
Additionally, personal protective equipment is an essential element in controlling exposure to
potentially hazardous biological material. It must be appropriately selected for the task, worn and
removed properly, and be in good condition (see section B9.1.3.2).
Probability of accident RG3 RG2 RG1
High High Medium Low
Medium High Medium Low
Low Medium Low Very low
Negligible Low Low Very low
B9.1.3.1
Biological Work Area Safety Plan
B9.1.3.1(a)
A laboratory-specific biological work area safety plan (Bio-WASP) is required for work with
biological materials at any containment level.
B9.1.3.1(b)
The bio-WASP will be reviewed as part of the IBC review/approval process.
B9.1.3.1(c)
The bio-WASP must be available & accessible in the work area.
B9.1.3.1(d)
The bio-WASP shall be reviewed at least biennially and updated necessary when new
biological materials, equipment, or methods are introduced.
B9.1.3.1(e)
The Biological Work Area Safety Plan (bio-WASP) should cover the items outlined below and be
specifically tailored to the needs of the individual work area and research.
i. General operations – Laboratory access and guidelines for workers/non-workers;
entry/exit procedures; procedures for identifying, reporting, and correcting problems; facility
procedures; pest management; general laboratory safety guidelines; lab notebook.
ii. Safety equipment and personal protective equipment – PPE requirements; proper
donning and doffing procedures for PPE; maintenance and decontamination of reusable
PPE; use, decontamination, and maintenance of equipment (BSC, autoclaves, eyewash,
showers, etc.).
iii. Emergency procedures – Accidents: spill, release, exposure, fire, etc.; documentation
and reporting requirements; terrorist threat; etc. See section B.7 for compliance
requirements and procedure guidelines.
iv. Training – Documented understanding of the bio-WASP manual; notification of the risks
associated with the materials to which workers may be exposed; frequency of in-lab
proficiency testing and other trainings/assessments; etc.
v. Practices and procedures – Safe handling, storage, and decontamination of biological
materials; handling of frozen samples; use of secondary containers; transporting samples;
aseptic technique and proper hand washing; management of sharps; waste handling and
disposal; facility decontamination and housekeeping; etc.
vi. Standard operation procedures (SOPs) are required for all laboratory functions (CHP,
A12.1) and must be reviewed and approved by the supervisor/PI (CHP, A12.2). Use of
images, pictures and flowcharts in SOPs is helpful. Written procedures should include the
information outline sections A12.1.1-A12.1.11 of the CHP.
vii. Health and medical monitoring – Signs/symptoms of exposure for all materials; fever
monitoring; OHP enrollment; vaccination requirements and or titer testing if required; etc.
viii. Biosecurity – Security measures taken to protect and control the use of biological
materials and research-related information (e.g., doors remaining locked, restricted access,
materials secured at the close of the workday, locking freezers, inventory and material
management procedures, adequate documentation, etc.); measures to assess personnel
are at the discretion of the PI, the granting agency, and/or the institutional Facility Security
Officer (in ORS).
Please contact EHS at 806-742-3876 or email lab safety at ehs.lab.safety@ttu.edu if you have
questions about bio-WASP content or for a review of your bio-WASP.
B9.1.3.2
Personal Protective Equipment (PPE)
Personal protective equipment includes all clothing and work accessories designed to serve as ‘the
last line of defense” against work hazards.
PPE should be dedicated to a specific work area and not moved among work areas; this is a
requirement for all areas operating at level 2 containment (i.e., ABSL2, BSL2, ACL2, etc.). If
you need PPE in another location (i.e., autoclave), carry it with outside surfaces folded
inward or place it on a cart.
Proper laboratory attire and PPE requirements for laboratory spaces are addressed in
section A4.3 of the CHP and shall be followed unless specific guidance pertaining to
biosafety is addressed in this section (B9.1.3.2), B9.2.2, or elsewhere in the biosafety
manual.
B9.1.3.2(a)
Gloves
Disposable gloves are commonly used in laboratories and are required for working with chemical
and biological materials. Double gloving may be recommended or required with certain materials
or procedures.
Nitrile gloves are recommended for working with chemicals; most glove manufacturers have done
performance test data for common chemicals. You are encouraged to research the protection
offered by your glove selection. Glove producers like Kimberly-Clark and Ansellpro offer glove
selection tools to help select proper gloves for your hazards.
Proper use of gloves includes the following:
i. Wash hands prior to donning gloves to remove lotions which may compromise glove
integrity.
ii. Glove cuffs should be worn OVER lab coat cuffs.
iii. When double gloving, the following tips can help:
a. Use different colors for inner and outer gloves.
b. Use extend-cuff gloves for the outer glove.
c. Turn the inner glove inside out to assist in donning the outer glove.
d. Use 1.5 inch blue painter’s tape to secure inner glove to lab coat or Tyvek sleeve.
Tab the end of the tape to facilitate easy removal of the inner glove. Taping the
inner glove facilitates easy removal of the outer glove as needed during work.
iv. Avoid touching your face, eyewear, personal items (e.g., cell phone) with your gloves.
v. Gloves shall be removed in such a way as to not contaminate the wearer or aerosolize
material on the gloves.
vi. Used disposable gloves shall be discarded immediately after removal and never reused.
vii. Gloves should be changed frequently and between procedures. Gloves shall be changed
when they are visibly contaminated and when integrity has been compromised.
viii. Frequent decontamination of gloves with 70% ethanol is encouraged in biological
applications.
ix. Gloves are not to be worn outside the workspace or common areas.
x. In general, gloves worn in biological laboratories are disposed of as biowaste.
xi. Hands should be washed anytime gloves are removed. Hands must be washed before
leaving the laboratory/work area. Camp sinks should be used for field work where facilities
are unavailable.
xii. Be aware of the use of laboratory chemicals that may breakdown a glove’s integrity (e.g.,
ethanol). Ethanol’s effect on glove integrity is generally not a concern unless other
chemicals which present a health hazard are being handled.
xiii. Specialty gloves (natural rubber, cryogenic, neoprene, thermal, PVC, Silver Shield®, etc.)
may be needed for certain procedures. Such PPE must be available, in good condition and
specified in the written procedure when required.
B9.1.3.2(b)
Eye and Face Protection
Proper use eye and face protection includes the following:
i. Body and eye protection must be worn when biological hazards are not behind a physical
barrier (e.g., closed cabinet, closed refrigerator, closed drawer, closed incubator, etc.).
ii. Regular prescription glasses are not a substitute for safety glasses.
iii. Over-the-glasses safety glasses should be available on-hand for workers who wear
glasses. Contact lenses may or may not be worn at the PIs discretion.
iv. The type of eye and face protection needed for a certain laboratory may vary with
procedure and must be ANSI-certified for the hazards encountered (i.e., impact,
splash/spray, UV light, lasers, etc.).
A brief description of common laboratory eye protection is below.
Safety glasses – Protective eyeglasses with impact-resistant lenses and safety frames constructed
of metal or plastic; primarily provide impact protection and should be used in conjunction with a
face shield or splash shield if splashes or sprays are likely; some provide UV protection.
Goggles – Tight-fitting eye protection that completely covers the eyes, eye sockets and the facial
area immediately surrounding the eyes. Some goggles may only offer impact-only protection and
are not designed to offer protection against splashes and spays. Splash goggles are specifically
designed to protect against splashes and sprays and must be worn when working with chemical /
biological agents or procedures that present a splash hazard.
Laser safety glasses/goggles – Provide protection from laser light; make sure your eye protection
addresses your laser class.
Face shields – Headgear providing a transparent sheet of plastic covering the entire face. They
protect against nuisance dusts and some splashes or sprays of hazardous liquids but may not
provide adequate protection against impact or splash hazards and are to be worn with eye
protection (i.e., safety glasses or goggles).
B9.1.3.2(c)
Body Protection
Different types of body protection (e.g., coats, coveralls, scrubs, gowns, and aprons) provide
different types of protection based on the design and material composition. Some may be particle
resistant, waterproof, flame resistant, and/or chemical resistant.
Proper use of body protection includes the following:
i. Lab coats must fall below the waist; knee-length is recommended.
ii. The lab coat selected shall address the hazards in the laboratory.
• A barrier coat is strongly recommended in containment level 2 work areas or for
those working in biosafety cabinets; this coat has knit cuffs and snaps up to the neck
to prevent exposure to potentially infectious materials.
iii. Laboratory coats should be comfortable and well-fitting; an improper fit may endanger the
user.
iv. Reusable body protection must be laundered when soiled and at regular intervals (e.g.,
monthly) to minimize contamination. Be sure the laundry service used is qualified to
manage the potential exposure to whatever may be present on the coats. Autoclaving or
bleaching coats prior to laundering is prudent but only necessary in the event of a spill or
other visible contamination.
v. Used laboratory coats shall not be stored in non-laboratory areas. Freshly laundered,
unused, laboratory coats may be stored in passageways if absolutely necessary.
vi. Body protection from containment level 2 work areas is work-area specific and shall not be
worn outside the work area or taken to other work areas. If needed for use of a communal
autoclave, they shall be transported on a cart with the materials to be autoclaved. Ideally a
designated coat is set aside for this purpose.
vii. Consider sleeve covers, protective suits, booties, aprons etc. as needed for hazards
associated with your work.
viii. Consult EHS if you have questions about PPE or if you feel respiratory protection is
needed.

B9.2 Working with Biological Materials

Federal and state regulations and guidelines govern laboratory safety to protect personnel, the
public, and the environment from biological hazards. Many granting agencies, including NIH,
require that grant recipients certify that they adhere to these guidelines and regulations. Failure to
provide evidence of adherence, if requested, can result in suspension and/or withdrawal of funds
from the University.
B9.2.1
The IBC Protocol Application
The principal investigator is responsible for the preparation and submission of the IBC Application
Form to the IBC for any project involving the items stated in B4.2.9 (restated below). Approval is
required before work begins in most cases.
Principle Investigators are required to submit work for IBC review for any of the following:
• Recombinant and/or synthetic nucleic acid (rNA/sNA) activities;
• Materials or agents potentially infectious to humans, plants, and/or animals – including the
storage or concentration of any potentially biohazardous materials;
• Except for general surveillance, arthropods that serve as vectors of disease to humans, plants,
or other animals, and arthropods that are considered an environmental hazard;
• Use of soil seed, plants, plant pathogens (e.g., bacteria, viruses, fungi, or parasites) or other
material received under a USDA APHIS compliance agreement or permit;
• Use of potentially infectious human and/or non-human primate materials;
• Use of cell lines that pose a danger to humans, animals, and/or plants, and/or those
immortalized by means that render them dangerous to humans, animals, and/or plants;
• The use of biologically derived toxins;
• Work involving prions or prion-like proteins;
• Any Select Agent or Toxin as listed in 7 CFR 331, 9 CFR 121, and/or 42 CFR 73.
• Necropsy of animals not under the care of the University Veterinarian; necropsy that includes
hands-on student involvement; and necropsy of animals with unknown health status and/or
animals reasonably suspected or known to be infectious;
• Other work as deemed necessary for review by the Biological Safety Officer; and/or
• Work with other potentially biohazardous materials conducted at, or sponsored by the
University for compliance with the guidelines in the CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories (BMBL) and approve those research projects that meet or exceed the
requirements in the BMBL.
The IBC protocol review is facilitated through the Cayuse Hazard Safety module. This is a
cloud-based platform for IBC protocol management and maintenance. No other form of
application or application amendment will be accepted.
EHS is available to consult and answer questions regarding form completion and any other
pertinent documentation prior to IBC review.
B9.2.1.1
Documents Required for Application Submission
The following documentation must be attached to your protocol in Cayuse:
(a) Safety plans (i.e., bio-WASP, field safety plan);
(b) Signed Personnel Assurance sheet for the personnel listed;
(c) State and/or federal permits for research materials on your protocol;
(d) Co-PI forms for Co-PIs listed on the protocol;
(e) Supporting documents such as SOPs, MOUs, or literature cited on your application; and
(f) OHP clearances from the occupational health provider (when required).
B9.2.1.2
Steps for Protocol Registration
These steps and links are available on the IBC Protocol Registration Page:
https://www.depts.ttu.edu/ehs/academicsafety/Biosafety/protocolregistration-bio.php.
(a) Register yourself and lab members to be listed on your protocol in Cayuse using the online
Cayuse Personnel Description Form:
https://www.depts.ttu.edu/ehs/academicsafety/Biosafety/ibccayuse-personnel.php.
(b) Complete and Submit your Protocol in Cayuse.
• A Researcher Toolbox has been created that has work aids for completing and
managing your protocol in Cayuse. The Toolbox is located at:
https://www.depts.ttu.edu/ehs/forms/TTUIBC_ResearcherToolbox.php
• You can view the status of your protocol at any time in Cayuse.
• Co-PIs and lab managers can create and amend a protocol on behalf of a PI, but only
the PI of record can submit the protocol.
(c) The Review Process.
• The PI submits the completed IBC application and attaches any associated documents
as those outlined in B9.2.1.1 within the Cayuse Hazard Safety module. These include:
the WASP, Other supporting documentation (i.e., SOPs, federal permits, literature,
etc.), OHP clearances, and Personnel Assurances and Co-PI Forms.
• The IBC Office may review the submission for completion and request changes prior to
forwarding the application to the committee.
• The protocol is distributed to the IBC committee members for review. Committee
member comments (if any) are collected by the IBC Office and sent back to the
submitting PI.
• If severe concerns are expressed by the committee or the protocol is called for full
committee review, the IBC may invite the PI to attend the upcoming meeting to discuss
their proposed work in-person.
B9.2.1.3
Conditions of Approval
(a) The laboratory spaces where work is to be conducted must meet the
biocontainment requirements for the work to be performed as determined by the
IBC.
(b) Requested clarifications or changes to the IBC Application by the IBC must be
addressed by the PI. The PI must either submit answers or a rebuttal to all committee
member comments no less than 5 days before the upcoming meeting to have the final review of their application on the meeting agenda. A list of meeting dates is on the IBC
homepage: https://www.depts.ttu.edu/ehs/academicsafety/icc/ibc.php.
(c) Protocols are discussed and approval granted at the monthly IBC meetings. In
special situations, approval may be granted outside of the formal meeting.
B9.2.1.4
Post-Approval Requirements
Print your protocol and associated attachments in hard copy and have lab members listed
on the document review. Maintain a copy of your current protocol in the work area. A
dedicated binder for this purpose will be provided by EHS at PI request.
B9.2.1.4(a)
Required Renewal and Annual Updates
PIs must submit an annual continuing review to the IBC and renew active protocols every
three years. If a protocol is completed prior to the continuing review or 3-year renewal period,
please contact the IBC Office by email at ibc.ehs@ttu.edu.
B9.2.1.5
Amending the IBC Application
All protocol amendments, including personnel amendments, are performed in Cayuse.
B9.2.1.5(a)
Amendments to the IBC Application are required when there are modifications to any of the
following:
i. PIs;
ii. Personnel;
iii. Research locations;
iv. Materials (e.g., vectors, gene targets, organisms, cell lines, tissues, disinfectants, etc.);
v. Methods; and /or
vi. When new hazards are introduced and not otherwise addressed in the protocol.
B9.2.1.5(b)
Amendments are subject to the conditions of approval in B9.2.1.3.
B9.2.1.5(c)
Certain amendments such as personnel and facilities are approval administratively through
the IBC Office. Changes to materials and methods are only administratively approved when
there is no change in the risks or objectives associated with the work previously approved
by the IBC. This is at the discretion of the Biosafety Officer or their proxy and may or may
not include the IBC Chair or Associate IBC Chair given the nature of the amendment.
B9.2.2
Licensing of a Lab Space
B9.2.2.1
The following is required for laboratory work to be performed using BSL1 containment.
Laboratories working at BSL1 must:
(a) A lockable door which remains closed at all times;
i. A self-closing door is ideal.
(b) Have a sink for handwashing;
(c) Have an eyewash;
(d) Have adequate illumination;
(e) Have screens for windows, or seal windows, when windows are present;
(f) Have an integrated pest management plan;
(g) Have appropriate signage on the door;
(h) Have access to a copy of the most current edition of the CDC document Biosafety in
Microbiological and Biomedical Laboratories (BMBL);
i. Electronic access must be independent of the internet and not tied to a specific
computer (i.e., flash drive or equivalent external storage device) that is accessible in
the lab.
(i) Have access to a copy of the most current edition of the NIH Guidelines for Research
Involving Recombinant DNA Molecules if a laboratory is doing rNA/sNA work;
i. Electronic access must be independent of the internet and not tied to a specific
computer (i.e., flash drive or equivalent external storage device) that is accessible in
the lab.
(j) Post emergency numbers in a visible location preferably on the door, near the phone or
both;
(k) Properly manage biowaste from generation to disposal (See section B8.3.);
(l) Have appropriate, labeled biological waste containers readily available (See section
B8.3.2.3.);
(m) Have waste segregation procedures and disposal plans in place for general waste, liquid
biological waste, solid biological waste, sharps, glass, etc.;
(n) Follow the practices outlined in B9.2.3.9 regarding the use of sharps;
i. An SOP guides the use and disposal of sharps in the work area.
ii. Mechanical means are readily available and used to replace blades on reusable
sharps.
(o) Use plasticware as a substitute for glass when possible;
(p) Have mechanical means to handle broken glass (i.e., tongs, forceps, dust broom, etc.);
(q) Have a hard copy of the current (bio)WASP available and accessible in the laboratory;
(r) Document worker understanding of the (bio)WASP and that they have been informed and
understand the hazards associated with their work;
(s) Have hard copies of EHS-required training certificates for all personnel;
i. A sign indicating the location of training records must be in the laboratory if these
records are stored in a different location.
(t) Have personnel complete rNA/sNA training when recombinant or synthetic nucleic acid
methodologies are used;
(u) Provide specific, in-laboratory training regarding duties, potential hazards, manipulations
of infectious agents, necessary precautions to minimize exposures, and hazard/exposure
evaluation procedures (e.g., physical hazards, splashes, aerosolization) are provided to
all personnel.
i. This training is documented, and proficiency level(s) are checked.
ii. Annual updates and additional training are required when equipment, procedures, or
policies change.
iii. EHS can work with the PI to facilitate this as needed.
(v) Put procedures in place to minimize the creation of aerosols;
(w) Post spill protocols;
(x) Don appropriate PPE at all times;
i. Gloves must be worn when working with biological materials or agents or may come
in contact with contaminated surfaces or equipment. (See B9.1.3.2(a) for specifics.)
ii. Long hair is secured.
iii. Skin below the waste is covered as appropriate for laboratory and field
environments.
iv. Solid shoes are worn.
v. Eye, face, and body protection is appropriate to the work being conducted. (See
B9.1.3.2(b) and B9.1.3.2(c) for specifics.)
(y) Wash hands before leaving the laboratory;
(z) Routinely decontaminate reusable PPE, the laboratory, and equipment within the
laboratory;
(aa) Immediately decontaminate surfaces, areas, and PPE after all splashes and spills;
(bb) Have a biological-specific spill kit and fresh disinfectant readily available at all times
while work is ongoing;
(cc) Not allow animals and plants that are not associated with the work being performed to
be present in the laboratory;
(dd) Use an overflow flask in-between suction flask and pump / building vacuum line when
suctioning potentially infectious substances;
(ee) Advise all persons entering the facility of the potential hazards, instruct them on the
appropriate safeguards, and require them to read and follow instructions on practices
and procedures.
(ff) Remove PPE in a manner that minimizes personal contamination and transfer of
infectious materials outside of the areas where infectious materials are manipulated.
(gg) Place all biological materials/agents in a durable, leak-proof container during collection,
handling, processing, storage, or transport to and within the laboratory.
(hh) Consumables and personal use products are kept outside of the work area (i.e., food,
drink, vapes, cosmetics, etc.).
(ii) Mouth pipetting is prohibited. Mechanical pipetting devices are used.
(jj) Cleaning equipment (i.e., brooms, mops, buckets, etc.) is dedicated to the work area.
When a biosafety cabinet (BSC) is present, the following applies:
(kk) Locate the BSC away from doors, room ventilation, heavily traveled areas, and other
disruptive equipment so as to not disturb airflow within the BSC;
(ll) Certify the BSC annually (i.e., within the past 12 months); and
i. If the BSC’s certification is expired, a sign is posted indicating the cabinet must not
be used.
ii. If the BSC has malfunctioned, a sign is posted indicating the cabinet must not be
used.
(mm) Use the BSC appropriately as outlined in B10.1.
When a laminar flow hood (clean bench) is present, the following applies:
(nn) Use a laminar flow hood appropriately as outlined in B10.2.
B9.2.2.2
The following is required in addition to B9.2.2.1 for a laboratory to be licensed as BSL2
space and maintain that laboratory designation.
Laboratories working at BSL2 must meet BSL1 (B9.2.2.1) and:
(a) Preferably locate a sink near the exit;
(b) Have self-closing doors;
(c) Have personnel complete Biological Safety training;
(d) Have personnel complete Bloodborne Pathogen training and OHP enrollment when
human materials are used;
i. OHP enrollment is facilitated by EHS
(e) Have restricted access to the lab;
i. Untrained persons who must enter the laboratory must be escorted at all times. In
general, minors, custodial staff, and maintenance workers are not allowed in the
laboratory. See section B9.2.4 for more details.
(f) Secure laboratory work, and decontaminate lab work surfaces at the end of each
workday;
(g) Evaluate the need for medical surveillance, OHP enrollment and immunization
requirements and implement as appropriate:
i. Dependent on materials or agents to be used.
ii. Evaluated in conjunction with IBC review.
(h) Place biohazard signage on lab equipment that contacts biological agents/materials;
(i) Maintain a log of the routine decontamination of laboratory equipment;
(j) Decontaminate the general laboratory area (including floor) weekly unless work variability
warrants a less-rigorous disinfection schedule as determined by the IBC.
(k) Conduct all aerosol-generating procedures that involve the manipulation of potential
infectious materials in a biosafety cabinet or other physical containment device when
possible.
i. In instances where a biosafety cabinet is not feasible, additional PPE may be used
as determined by the risk assessment done in conjunction with IBC review.
(l) Incorporate an in-line HEPA filter between the overflow flask and vacuum source when
suctioning potentially infectious substances.
(m) Test PI-owned autoclaves within the laboratory that are used for biowaste at the
applicable frequency described in B8.6.4.6(a).
B9.2.2.3
Prior to BSL2 work, a survey is needed to confirm a work area meets the criteria for BSL2.
If you have questions about these requirements, please contact EHS at 806-742-3876.
After inspection, once all criteria are met, a sign designating the lab space as BSL2 will be
provided and affixed to the outside door by EHS.
B9.2.2.4
New laboratories/work areas must be commissioned prior to work being conducted
regardless of the containment level.
B9.2.3
Special Considerations for Certain Research Foci
B9.2.3.1
Animal Use Considerations
The following is required for animal work to be performed using ABSL1 containment.
Laboratories working at ABSL1 must meet BSL1 and:
(a) Have exterior doors that are self-closing;
(b) Have exterior doors that are self-locking;
(c) Have windows that are resistant to breakage;
(d) Have a sink for handwashing located at the exit of are areas where infectious agents are
handled or where animals are housed;
(e) Have a safety shower;
(f) Have internal facility fixtures (e.g., light features, air ducts, and utility pipes) and
penetrations in floors, walls, and ceiling surfaces (including openings around ducts, doors,
doorframes, outlets, and switch plates) installed to facilitate cleaning and minimize
accumulation of debris and fomites;
(g) Have interior surfaces that are water, slip, and chemical resistant;
(h) Have floors with drains sloped toward the drain;
(i) Have traps on drains filled with water, appropriate disinfectant, or sealed;
(j) Have ventilation that meet the standards of the Guide for the Care and Use of Laboratory
Animals;
(k) Have ventilation systems able to withstand high heat and moisture loads produced from
cleaning;
(l) Have the facility maintained to facilitate cleaning and housekeeping;
(m) Have furniture minimized and able to withstand anticipated loads and uses;
(n) Maintain a log of access to the facility;
(o) Include animal specific policies regarding biosafety, biosecurity, emergencies and animal
disposition during emergencies in the (bio)WASP;
(p) Have medical staff informed of the animal specific occupation hazards within the facility
involving research, animal husbandry, care, and manipulation;
(q) Include animal allergy prevention as a part of medical surveillance;
(r) Have PPE (i.e., gowns, uniforms, scrubs, laboratory coats, or other PPE) available in
areas where animals are housed or animal manipulation areas;
(s) Have eye protection disposed of with other contaminated waste or decontaminated after
each use;
(t) Not allow protective outer clothing to be worn outside of areas where infectious materials
and/or animals are housed of manipulated;
(u) Not allow gowns and uniforms to be worn outside of the facility;
(v) Have the final rinse temperature of cage washers be 180 °F;
(w) Have PPE that is appropriate for large animal use;
(x) Have bite or scratch resistant gloves for animal work when appropriate;
(y) Remove or recap needles following approved methods;
(z) Have male and female animals separated with double barriers or measures to prevent
reproductive transmission except where needed for experimentation;
(aa) Have genetically engineered neonates permanently marked within 72 hours of birth;
(bb) Have transgenic animals contain distinct, biochemically stable, and assayable DNA
sequences for identification from among non-transgenic animals;
B9.2.3.2
The following is required for animal work to be performed using ABSL2 or ABSL2-Ag
containment.
Laboratories working at ABSL2 must meet BSL2 and:
(a) Have a sink for handwashing available at the exit of each segregated area, if the
segregated area is where infectious agents or animals are manipulated or housed;
(b) Have an autoclave available;
(c) Have inward airflow;
(d) Discharge exhaust air outside without recirculation to other rooms;
(e) Have ventilated cage systems designed for microorganisms containment;
(f) Have HEPA filters and housing certified annually on actively ventilated cage systems;
(g) Have sealed exhaust plenums, and failure alarms for actively ventilated cage systems;
(h) Have primary containment equipment appropriate for the animal species in containment,
such as solid walls and bottoms in cages or housing areas, or microisolator lids;
(i) Transport biological materials from animal containment in non-breakable, sealed primary
containers which are enclosed in non-breakable, sealed secondary containers;
(j) Disinfect all transport containers prior to removal from the facility;
(k) Decontaminate cages prior to washing where infected animals were housed;
(l) Use alkaline digestion or incineration for decontamination and disposal of carcasses;
(m) Maintain a log of decontamination process routine verification;
In addition to ABSL2, areas working at ABSL2-Ag must:
(a) Have entrances through a series of barriers or procedures that provide a distinct
separation between containment and non-containment areas;
(b) Have a boot wash at the entry/exit of the animal room or containment barrier;
(c) Replace boot wash disinfectant solution according to the manufacturer’s instructions;
(d) Have floors, ceilings, and walls of animal rooms made from monolithic materials that are
durable and resistant to disinfection which may include pressurized sprays, hot water,
steam, or chemicals;
(e) Have air handling services where large animals are housed or manipulated that are
capable of minimizing emissions and particulates in addition to meeting animal welfare
requirements;
(f) Have electrical wiring and equipment that are installed in wet or hazardous locations
sealed and grounded with animal welfare considered;
(g) Have pens, gates, and/or animal restraint systems appropriate for the animal species with
consultation from veterinary staff;
(h) Have equipment that is sealed or coated such that it is resistant to disinfection and
cleaning or is completely disassembled for complete decontamination;
(i) Have equipment that is free of pinch points and sharp edges that could injure animals or
personnel;
(j) Have equipment for cold storage and decontamination of large animal carcasses and
solid/liquid waste;
(k) Have policies integrated into the (bio)WASP to limit contact between containment staff
and susceptible animals to the outside of the containment area;
(l) Have all staff trained to handle time-sensitive emergencies involving personnel pinned or
entangled by animals or equipment;
(m) Establish systems for a minimum of two workers to be present in the containment area at
all times (i.e., a “buddy system”), or have another means of monitoring worker safety in
containment.
(n) Have agent or infected animal manipulations performed exclusively in a BSC, or if not
possible due to animal size limitations, another primary containment device;
(o) Have contaminated effluent collected for decontamination and validated for inactivation
before discharge into drainage systems;
B9.2.3.3
Arthropod Use Considerations
The following is required for arthropod work to be performed using Arthropod Containment
Level 1 (ACL1).
Laboratories working at ACL1 must meet BSL1 and:
(a) Have exterior doors locked at all times;
(b) Have door openings covered (i.e., rigid panels, glass, screens, plastic sheets, or cloth) so
that they minimize the escape and entrance of arthropods;
(c) Have windows that prevent escape by smallest of arthropods contained within as well as
prevent the entry of wild arthropods;
(d) Have interior walls that are light colored;
(e) Have interior floors that are light colored, smooth, and uncovered;
(f) Have electrical boxes, lighting, switches, wiring, conduit, and other penetrations sealed
with caulk, foam, or other appropriate material able to withstand disinfection;
(g) Have work surfaces and laboratory furniture that are white or light gray, water resistant,
impervious to arthropods, and resistant to disinfection and heat;
(h) Have benches, cabinets, and equipment easy to inspect for escapees;
(i) Locate furniture and incubators containing arthropods such that accidental contact and
release are minimized (i.e., out of the flow of general traffic, avoiding hallways, or in
closets);
(j) Reduce harborage and breeding areas;
(k) Maintain a log of escapees;
i. If exotic arthropods are in use, maintain a log of exterior escapees;
ii. If escapees are found, review work practices before continuing work;
(l) Wear clothing that minimizes exposed skin;
(m) Wear laboratory coats, gowns, and uniforms that are white;
(n) Not allow the entry of overcoats, hats, purses, or other personal items into the
containment area;
(o) Have cages that hold arthropods effective at preventing the escape of all life stages;
(p) Label containers and cages that hold arthropods with the species, strain/origin, date of
collection, and responsible investigator;
(q) Clean cages and other culture containers to prevent arthropod survival and escape (e.g.,
heated to, or chilled below, lethal temperature);
(r) Kill all life stages of arthropods with hot water or freezing before disposal;
B9.2.3.4
The following is required for arthropod work to be performed using Arthropod Containment
Level 2 (ACL2).
Laboratories working at ACL2 must meet BSL2 and:
(a) Have doors that have physical barriers (i.e. overlapping sheets and screens) or air
curtains;
(b) Have a contiguous double-door vestibule that prevents flying and crawling arthropods
from escaping as the entrance;
(c) Have the doors of the entrance not opened simultaneously;
(d) Have doors that are windowless, or block the windows of interior doors, seal the windows,
and use break resistant materials;
(e) Have internal doors that open outward or are sliding, and are closed at all times when
arthropods are present;
(f) Have downdraft fans in the vestibule and internal corridor to help prevent flying arthropod
escape;
(g) Have ceilings that are as low as possible;
(h) Minimize light fixtures, pipes, ducting, electrical boxes, switches, wiring, conduit, and other
penetrations and seal them with caulk, foam, or other appropriate material able to
withstand disinfection;
(i) Have light fixtures that are flush to the ceiling;
(j) Eliminate harborage and breeding areas;
(k) Have equipment that may store or accumulate water, such as a humidifier, screened or
chemically treated to prevent arthropod access or breeding;
(l) Have a negative pressure gradient into the containment area;
(m) Have ventilation systems with filters or barriers that prevent the escape of arthropods;
(n) Have vacuum systems with filters or barriers that prevent the escape of arthropods;
(o) Have floor drains and other plumbing features covered by appropriately sized mesh or
filters to prevent arthropod escape;
(p) Have a communication and/or a computer system that allows for communication and data
transfer between the interior and exterior of the facility;
(q) Have an autoclave available in rooms where arthropods are present;
(r) Maintain an annual log of facility inspections to ensure maintenance and alterations have
not compromised containment;
(s) Maintain a logbook of visitors;
(t) Use vacuum devices dedicated to a single facility;
(u) Manipulate non-flying arthropods in a dedicated area within a BSL2 space using a moat
system if a dedicated insectary is not available;
(v) Store all supplies for insect maintenance in a designated area in cabinets or drawers that
are tight fitting;
(w) Store arthropod diet in sealed containers;
(x) Have cages that are shatter-proof and screened to prevent escape during holding,
removal, and introduction;
(y) Transfer arthropods between holding and manipulation areas in non-breakable, secure
containers;
(z) Have cages or containers clearly labeled to distinguish infected and uninfected
arthropods;
(aa) Have all infectious or potentially infectious samples labelled;
(bb) Have infected or potentially infected arthropods autoclaved after being rendered nonviable;
(cc) Open packages with foreign sourced materials in a BSC;
(dd) Have vector packing materials autoclaved immediately after removal of cultures and
specimen from the package;
B9.2.3.5
Plant Research
Permits from the USDA must be obtained for the inter- and intrastate movement of certain plant
and soil materials.
The following is required for plant work to be performed using Plant Containment Level 1
(PCL1).
Laboratories working at PCL1 must meet BSL1 and:
(a) Adhere to all USDA-PPQ requirements;
(b) Sterilize or disinfect all incoming material to prevent entrance of external vectors;
(c) Not allow overcoats, hats, backpacks, purses and other personal objects in the
containment areas;
(d) Maintain a log of in progress experiments;
For PCL1 work in greenhouses:
(e) Have doors between the greenhouse and the facility that close completely and seal to
their frames;
(f) Have audible alarms on emergency exits that activate when opened;
(g) Have floors constructed of materials impervious to the organisms and that can withstand
repeated disinfection;
(h) Have translucent materials strong enough to guarantee security and frames to support the
walls and ceilings;
(i) Have weatherproof electrical boxes, receptacles, light fixtures, and switches;
(j) Maintain a log of leak testing;
B9.2.3.6
The following is required for plant work to be performed using Plant Containment Level 2
(PCL2).
Laboratories working at PCL2 must meet BSL2 and:
(a) Have exterior doors locked at all times;
(b) Have windows, penetrations, and translucent panels sealed to the frame with caulk or
other appropriate materials;
(c) Not allow drop in ceilings when BSCs are not used for containment;
(d) Have connections in air ducts, plenums, and vents sealed with caulk or other appropriate
materials;
(e) Have outlets and inlets (i.e., external exhaust, outside air intake, and internal air diffusors,
sewer drains, traps & vents, etc.) filtered with appropriately sized mesh or filters;
(f) Have HEPA filters changed annually or when the maximum resistance is met, and have
them recertified annually or when they are changed;
(g) Have an autoclave available;
(h) Use a BSC to work with pathogenic bacteria, nematodes, fungi, and viruses;
(i) Sterilize filters before disposal;
(j) Autoclave or incinerate contaminated organisms as soon as they are detected, even if that
requires destruction of a beneficial culture;
(k) Autoclave or incinerate packing materials after the removal of specimen and cultures;
(l) Autoclave laboratory coats, coveralls, and other clothing before removal from the facility;
(m) Not allow offices within containment areas;
(n) Open foreign sources packages in a BSC or in one enclosed space that is easy to
disinfect;
(o) Maintain a log of experimental plants, microorganisms, and/ or animals when they are
brought into the facility or removed;
For PCL2 work in greenhouses:
(p) Use one primary entry/exit;
(q) If detached from the main facility, have interlocking vestibules at each door with 6 feet
between the doors from threshold to threshold;
(r) Emergency doors are not used as an entrance, have external handles removed, and have
internal hinges;
(s) Have airlock, directional airflow, filtration, and ventilation that prevent organism escape from
containment;
(t) Have electrical boxes, lighting, switches, wiring, and conduit sealed with caulk, foam, or
other appropriate material capable of withstanding disinfection;
(u) Have a communication system between the interior and exterior of the facility to prevent
organism escape from containment;
B9.2.3.7
Collection and Manipulation of Human Material
Resources used as standards for the collection and manipulation of human materials:
CLSI GP41: Collection of Diagnostic Venous Blood Specimens
CLSI GP44-A4: Procedures for the Handling and Processing of Blood Specimens for Common
Laboratory Tests; Approved Guideline – 4th Ed.
WHO guidelines on drawing blood: best practices in phlebotomy
AABB: Fundamental Standards for Blood Collection and Transfusion 1st Ed.
B9.2.3.7(a)
The reuse of “stress ball” style squeezing devices is not allowed for blood collection. Use a
racquet ball or other device that can easily be decontaminated.
B9.2.3.8
Basics to Aseptic Technique (reference EHS SOP 3.2)
Proper aseptic practices are an essential tool in biological research.
Aseptic techniques will vary by laboratory and task, only general concepts are addressed below.
(a) Zone workspaces within the area from clean to dirty, with the most contaminated or risky
work being performed as far away from egress locations as possible. Perform sensitive
tasks that do not involve biological materials in a laminar flow hood (i.e., media prep,
equipment assembly, etc.).
(b) Manipulation of biological material should be done in a BSC when feasible, this is
especially important when force is applied to a material as aerosols may be produced.
(c) Use laminar hoods and BSCs correctly. Misuse can contaminate you, your product, and
the environment.
(d) Keep work surfaces uncluttered and free of non-essential materials.
(e) Arrange workflow from clean to dirty to avoid passing contaminated material over noncontaminated material.
(f) Decontaminate work area before beginning work.
(g) Regularly decontaminate equipment.
(h) Change gloves frequently and practice good hand hygiene. Disinfect the outer surface of
gloves with 70% ethanol or other disinfectant regularly.
(i) Wear proper, well-fitting PPE. Lab coat/gown shall be buttoned all the way and glove
cuffs should cover coat or gown cuffs.
(j) Work vigilantly and deliberately when handling materials.
(k) Work near a proper flame when working on an open bench top, if work requires. A proper
flame has a bright, inner pale blue cone surrounded by a more translucent outer cone.
(l) Avoid placing caps/covers on the work surface, and when you must, place them face
down on the work surface.
(m) Use tools (forceps, tube openers, etc.) to open and close tubes containing biological
material and decontaminate them between samples.
(n) Clean up spills appropriately and immediately.
B9.2.3.9
Proper Use of Sharps (reference EHS SOP 4.1)
Disposal of sharps is in section B8.8.1.
(a) Avoid using sharps whenever possible; use blunted needles and plastics instead.
(b) Use devices with safety features to isolate sharps and be familiar with function and
features prior to use.
(c) Use disposable sharps instead of reusable sharps to avoid hazards associated with
decontaminating the items.
(d) Be prepared to use the device the moment the sharp is exposed and secure or discard
immediately after use.
(e) Stabilize hands or use a one-handed technique when uncapping devices to prevent recoil
injuries.
(f) When drawing solutions from a vial, immobilize the syringe on the hypothenar eminence
located at the base of the hand and insert the vial on the needle.
(g) Do not bend, break, or otherwise manipulate needles by hand.
(h) Do not remove needles. Discard needles and syringes as an intact unit immediately after
use into an FDA-approved sharps container.
(i) Do not recap needles or other sharps. If needles must be recapped a valid written reason
for recapping, as well as a protocol must be submitted to and approved by EHS.
Appropriate documented training must be given to each individual and a documented
demonstration of proficiency must be recorded.
(j) Use care and caution when cleaning up after procedures that require the use of sharps.
(k) Reusable sharps must be placed in a closable, rigid container for transport,
decontamination, and storage. The sharp end of items must be secured in such a manner
as to prevent accidental injury.
(l) Sharps containers shall be immediately accessible. Locate sharps containers in areas in
which needles are commonly used.
(m) Do not hand-pass exposed sharps. Place sharps in a predetermined neutral zone to pass
objects to others.
(n) Do not overfill sharps containers. Remove from service when at the fill line (e.g., 3/4 full),
close, autoclave if desired, and dispose of in accordance with University Operating Policy
60.10.
In the event of a needle stick or other sharps-related injury: Expose the wound; express the wound,
cleanse the wound, and then bandage the wound. Notify the PI and seek follow up treatment as
needed. An incident report must be filed with EHS within 24 hours of the event.
Access to the online form for Risk Management is available on the EHS website at:
http://www.depts.ttu.edu/ehs/about/incident-reporting.php.
Near misses should be reported to EHS by submitting a SCAN form on the EHS website
https://www.depts.ttu.edu/ehs/about/scan.php.
B9.2.4
Entry Procedures for Non-Laboratory Personnel into Containment Level 2 Laboratories
As described in section B9.2.2.2(e), only authorized personnel are allowed in Containment Level 2
laboratory spaces; however, at times, entry of non-laboratory personnel may be necessary.
B9.2.4.1
Occasions of Entry by Custodial Personnel
Custodial Services can be reached during university business hours at 806-742-9777. A red dot
will be placed on the door by custodial staff as a signal not to enter the laboratory space. This is a
requirement for Containment Level 2.
B9.2.4.1(a)
Once a laboratory is designated as containment level 2 the PI or laboratory manager must
notify custodial services that they are not to enter the laboratory.
B9.2.4.1(b)
General laboratory wastes (non-contaminated waste) shall be placed outside the laboratory
for custodial pick up.
B9.2.4.1(c)
Arrangements can be made with custodial for semiannual/annual floor waxing.
The laboratory must be properly disinfected prior to custodial entry, all chemical and
biohazardous materials must be stored.
While in the laboratory, custodial personnel must:
i. Don clean PPE upon entry into the laboratory; PPE is supplied by the PI.
ii. Be escorted by laboratory personnel at all times.
iii. Wash their hands prior to leaving the laboratory.
B9.2.4.2
Occasions of Entry by Maintenance Personnel
B9.2.4.2(a)
If maintenance workers must enter a laboratory space, they first must notify the PI or
designated emergency contact. Experiments in progress should be secured and suspended if
possible.
B9.2.4.2(b)
While in the laboratory, maintenance personnel must:
iv. Don clean PPE upon entry into the laboratory; PPE is supplied by the PI.
v. Be escorted by laboratory personnel at all times.
vi. Wash their hands prior to leaving the laboratory.
B9.2.4(c)
Should maintenance workers enter a lab space for emergency purposes outside of business hours,
they must notify the PI or emergency contact prior to entry and still follow B9.2.4.2(b).
B9.2.4.3
Occasions of Entry by Others
Entry into laboratory areas by non-laboratory personnel is addressed in A6 of the University
Laboratory Safety Manual. The expectations for notification and communications of visitors to the
lab should be outlined in the bioWASP or IBC protocol. The following outline additional
requirements for Containment Level 2 research areas:
(a) Visitors must be escorted at all times by lab personnel with the approval of the PI.
(b) Visitors shall be notified of the hazards present in the laboratory and wear the same PPE as
workers are required to as stated in A6.2. If PPE is refused or clean PPE is unavailable,
then entry into the laboratory shall be denied.
(c) Hands must be washed after PPE is removed, prior to leaving the laboratory.
(d) Minors under the age of 16 are not permitted in Containment Level 2 spaces under any
circumstances.
(e) Minors ages 16-17 may work in accordance with the specifications of section A8.3.1 of the
University Laboratory Safety Manual unless the building or laboratory-specific manual is
more restrictive.

B10 COMMON LABORATORY EQUIPMENT USEAGE

SOPs are required for all laboratory equipment. See LSM A12.1.

B10.1 Biosafety Cabinets

The biosafety cabinet (BSC) is designed to provide protection to the user and the environment
when appropriate practices and procedures are followed; certain BSC classes also protect the
materials within the cabinet. The three types of BSCs (Class I, II, III) are described in Appendix BD.
The common element to all classes of biosafety cabinets is the high efficiency particulate air
(HEPA) filter. This filter removes particulates of 0.3 microns with an efficiency of 99.97%; however,
particles both larger and smaller than 0.3 microns are removed with increased efficiency.
B10.1.1
Chemical and Radioactive Materials in the BSC
Based on the unit’s design, work with chemicals or radioactive materials may require
special measures, or such work may be prohibited within the BSC. EHS approval is needed
before work with chemical or radioactive materials occurs within the BSC.
While the HEPA filter can protect the worker from most biological hazards when used properly, it
does not remove radiation, hazardous chemicals, vapors, or gases. A brief outline of common
limitations according to the BMBL is below. If you have questions regarding what type of BSC you
have or if your work can be done safely within your BSC, please contact EHS at 806-742-3876.
1. Flammable or otherwise volatile chemicals are not used in a Class II, Type A BSC. Limit
the use of ethanol to decontamination of gloves and surface decontamination of the BSC.
This cabinet generally exhausts HEPA filtered air back into the workspace and recirculates
the remainder. The electrical systems of Class II BSCs are not spark-proof. Thus, this
practice creates an avoidable hazardous situation which may result in fire or personnel
exposure to toxic chemical vapors; however, many liquid chemicals, including nonvolatile
antineoplastic agents and chemotherapeutic drugs can be safely handled within Class II,
type A and B BSCs.
2. BSCs should not be used for labeling biohazardous materials with radioactive isotopes.
Hard-ducted, ventilated containment devices incorporating both HEPA and charcoal filters
in the exhaust systems are necessary for the conduct of this type of work.
3. Work with chemical carcinogens and other toxic substances within a Class II BSC requires
additional treatment measures for the exhausted air. Careful evaluation must be made of
potential problems associated with decontaminating the cabinet and the exhaust system in
this case. Air treatment systems, such as a charcoal filter in a bag-in/bag-out housing may
be required so that discharged air meets applicable emission regulations.
For these reasons, a chemical fume hood is to be used for procedures using volatile, toxic
or carcinogenic chemicals.
B10.1.2
Biological Materials
Certain biological materials and procedures may require the use of a BSC. The requirement
for a BSC at BSL2 is determined by a risk assessment during IBC review.
A biosafety cabinet should be used to protect you and the environment in the following
conditions:
1. When performing procedures with a high potential for creating aerosols or those that might
cause splashing, spraying or splattering of droplets of biological materials.
(a) Any procedure that imparts energy to a liquid sample or microbial suspension such as
centrifuging, grinding, blending, vortexing, sonicating, vigorous shaking/mixing/pipetting,
opening containers of materials whose internal pressures may be different from ambient
pressures (e.g., cryovials, lyophilized samples, fermenters, etc.), and inoculation and
tissue harvest procedures involving inoculated animals.
2. When large quantities or high concentrations of organisms are used.
3. For processing of human materials or samples where disease status is unknown.
This is not an exhaustive list, only a collection of general guidelines. When in doubt, use the BSC
or call EHS with your questions 806-742-3876.
B10.1.3
Handling and manipulation of BSL3 agents is ALWAYS performed in a BSC. Additional
PPE, including respiratory protection, may also be required.
B10.1.4
Installation, Maintenance and Certification
1. BSCs should be installed away from the lab entry in an area with restricted foot traffic and
little equipment which creates air movement that may disrupt the protective air curtain of the
BSC. The overall air balance of the laboratory and adequate clearance for air intake, work,
and ducting are also considerations when selecting your cabinet and its location. Consult
EHS for assistance in determining your BSC’s location.
2. Biosafety cabinets are highly customizable; however, certain features are prohibited or not
recommended.
(a) Sinks are not allowed in BSCs.
(b) Ultraviolet (UV) lamps are not recommended in BSCs. If installed, lamps must be
properly maintained and never used as the sole source of decontamination.
• Check manufacturer guidelines for maintenance and frequency of replacement. BSC
UV lamps must be properly maintained and never used as the sole source of hood
decontamination.
General maintenance includes:
i. Cleaning bulbs weekly to remove dust and debris which may lessen effectiveness of
UV light.
ii. Checking bulb regularly with a UV meter to ensure a germicidal intensity of UV light
is being emitted.
iii. Closing the sash when the lamp is on and turning off the lamp when room is
occupied.
3. Biosafety cabinets require regular maintenance and certification by a professional
technician to assure that it protects you, your experiments, and the environment.
(a) Each cabinet must be certified when it is installed and annually after installation.
(b) Moving the cabinet or repairs made to the cabinet void any current certification such that
if the cabinet is moved from the original place it was installed and certified, or if any
repairs are made, recertification is required before use is resumed.
(c) If a cabinet needs repairs to plenums of the unit, it must be fumigated by a third party
prior to repairs.
(d) Switches and repairs of that nature can be completed in-house by qualified lab
personnel, a third party, or the physical plant so long as the BSC plenums are not
accessed or compromised.
4. Annual BSC certification is the PI’s responsibility to coordinate and is completed by a third
party (not TTU-EHS); please call EHS if you need a list of vendors.
Call EHS for assistance with cabinet selection and installation guidance.
B10.1.5
Guidelines for Operation of a Class II Biosafety Cabinets
Proper PPE is required while working in the BSC. At minimum, a fastened lab coat, gloves,
and safety glasses are required. Glove cuffs should cover jacket cuffs, especially when handling
sensitive materials or potentially infectious agents. Additional PPE requirements are necessary for
handling and manipulation of BSL3 agents.
B10.1.5.1
Preparation
(a) Prepare a checklist to minimize arm movements in and out of the BSC which disrupts the
delicate protective air curtain.
(b) Check certification date on BSC. Certification must be within the past 12 months. If
certification has expired, DO NOT USE THE BSC.
(c) Turn on fluorescent light and turn off the UV light if in use.
(d) Ensure the sash is in the appropriate position. Turn on the blower fan. If blower was off,
allow at least 5 minutes before beginning cabinet disinfection.
(e) Compare the pressure reading on the magnehelic gage to the certification sticker. If the
gage reads at or within 10% greater pressure than the inspected value, the BSC may be
used. If the gage reads lower than the inspected reading or more than 10% greater, then
do NOT use the BSC. Post an out of order sign and call a vendor for repairs.
(f) Check gauges/monitors to ensure the unit is functioning properly. Do not work in a BSC
while a warning light or alarm is signaling.
(g) Disinfect the interior surface of the sash, interior walls, then work surface with a disinfectant
determined by the risk assessment to address the particular agent.
i. Do not raise the sash beyond operable height.
ii. Ethanol may not be adequate as the sole disinfectant for the interior surfaces of the
BSC. Contact time is abbreviated within the cabinet while the blower is running thus
appropriate contact time may not be met.
iii. Bleach should be used with caution to disinfect BSCs given it’s corrosivity. If used,
follow with an ethanol or sterile water rinse to avoid corrosion of the stainless-steel.
(h) Plastic-backed absorbent liners may be used so long as they do not obstruct the front or
rear grille openings. Use of this material facilitates clean up and reduces spatter in the event
of a spill. Liners must be decontaminated or placed in a closed autoclave bag within the
BSC before removal from the BSC. It can be folded – absorbent side together, plastic
backing out - and placed in a biowaste bin after a spill or when work is complete.
(i) Disinfect the surfaces of all materials to be placed in the cabinet with 70% ethanol or other
disinfectant – allow adequate contact time. Only the items necessary for the immediate
work should be placed in the BSC.
i. Keep the front and rear grills clear. Place materials on metal risers or shelves to
help reduce disturbance to the grill area.
ii. Arrange workflow should be clean to dirty.
iii. Extra supplies (gloves, extra tips, etc.) should be stored outside the BSC. Keep the
work area free of unnecessary equipment/supplies which may affect proper airflow
and subsequently, your protection.
iv. Bulky items should be placed to the rear and to one side of the work surface.
v. Place supplies, especially aerosol-generating equipment such as vortexes, as far
back as possible in the cabinet.
vi. Biowaste must be contained or decontaminated within the cabinet. Movement of
hands in and out of the cabinet to discard pipettes and other materials creates
turbulence that disrupts the air barrier which maintains sterility inside the cabinet and
protects the worker. Taking exposed contaminated materials outside the BSC
unnecessarily introduces contamination to the environment outside the BSC. Only
horizontal pipette discard trays should be used. A shallow pan filled with a
disinfectant suitable for biowaste treatment (i.e., not ethanol) is a good option.
Contact EHS if you have questions.
vii. Include disinfectant and paper towels in your supply list to manage spills quickly if
they occur.
(j) Use a surface-decontaminated tub to move materials in groups into and out of the BSC to
minimize disruptions to the air curtain.
(k) Locate liquid waste traps with disinfectant inside the cabinet and use a hydrophobic filter to
protect the vacuum lines. If traps must be located on the floor outside the BSC, place them
in a secondary container to prevent spilling.
(l) Place the sash at the proper height for operation. Keep your head out of the hood; adjust
your chair so that your face is above the sash opening. The BSC is now ready for biological
materials.
B10.1.5.2
Working in the BSC
(a) Once arms are in the BSC, delay work by 1 min to allow the air curtain to stabilize.
(b) Work as far to the back (beyond the grille) of the BSC workspace as possible.
i. Work at least 6 inches beyond the front grille.
ii. Move smoothly and deliberately in and out of the BSC, perpendicularly to the unit –
avoid side-to-side, sweeping movements.
iii. Do not rest your arms on front grille.
(c) Always use good aseptic and microbial technique when working in a BSC.
i. Use mechanical pipetting aids.
ii. Keeping clean materials away from aerosol-generating activities to minimize cross
contamination.
iii. Work with sensitive materials in the air split.
iv. Keep open tubes/bottles in a vertical position and do not place lids on the work surface.
v. Hold the lid above the sterile surface of petri or tissue culture dishes.
vi. Recap or recover items as soon as possible.
(d) The agent is the last item added to the BSC.
(e) Remove outer glove if double gloving or decontaminate gloves before entry into and
removal from the BSC.
(f) Open flames shall not be used inside a BSC. Flames disrupt the airflow and contribute to
the heat load inside the BSC. Flames have also burned holes through HEPA filters and
caused explosions in BSCs.
(g) Disposable sterile loops/needles should be used in place of metal loops or needles to avoid
incinerator or bead bath use.
(h) Follow spill protocols in the event of a spill (see section B7.2.4). Leave the BSC running
while you are cleaning up the spill. If you are uncertain on how to manage a spill, please
call EHS for assistance at 806-742-3876.
(i) When work is completed with the BSC still running, first contain, surface disinfect, and
remove the agent. Contain and remove the agent, then proceed to decontaminate the
surfaces of supplies and equipment and remove them from the cabinet – working clean to
dirty and using the tub in-tub out method. Wipe down the interior surface of the sash,
interior walls, and the work area with a disinfectant determined by the risk assessment.
Remove all materials from the BSC (except for pipettors if you choose). Close the sash if
desired and turn off the BSC unless laboratory protocol states the BSC is to be kept
running.
(j) If a UV light is used in the BSC, it may be turned on at this point if others are not working
nearby. UV radiation shall not take the place of chemical disinfection in the cabinet interior.

B10.2 Laminar Flow Hood

A laminar flow hood or "clean air bench" is not a BSC. In these units, HEPA-filtered air is
discharged horizontally across the work surface and toward the user, or vertically, downward onto
the work surface ultimately exposing the user to the contents. Laminar hoods only provide product
protection. They can be used for certain clean activities, such as dust-free assembly of sterile
equipment or electronic devices, media dispensing, and most nucleic acid work.
Laminar hoods do not require annual certification; however, EHS recommends they be certified
annually to protect the integrity of your work.
B10.2.1
Laminar hoods shall never be used as a substitute for a BSC. The following materials are
not to be used in laminar flow hoods:
1. Flames;
2. Any viable organism / material including cells for cell/tissue culture;
3. rNA/sNA work if the host-vector system is classified as RG2, is self-replicating, or has the
capability to insert itself into DNA; and/or
4. with any other potentially infectious / hazardous materials.

B10.3 Centrifuge Containment and Safety

B10.3.1
When centrifuging materials:
1. Examine centrifuge tubes and bottles for cracks or stress marks before using them;
2. Use the proper size of rotor or bucket for your tubes/bottles/plates;
3. Never overfill centrifuge tubes since leakage may occur when tubes are filled to capacity;
4. Fill centrifuge tubes no more than 3/4 full; and
5. Use a tube/plate/bottle with an equivalent amount of water as a balance when odd numbers
of items are centrifuged.
B10.3.2
Centrifuge safety buckets and sealed rotors protect against the release of aerosols and are
required for the centrifugation of RG2 material.
B10.3.3
When centrifuging potentially infectious material, delay the opening of the centrifuge at
least 5 minutes or open safety buckets/rotor within a BSC.

B10.4 Vacuum Lines

B10.4.1
All vacuum lines used to aspirate supernatants, tissue culture media and other liquids that
may contain potentially infectious or genetically modified materials must be protected from
contamination using a collection flask and overflow flask containing a disinfectant.
B10.4.1.1
Collection and Overflow Flasks
Collection tubes should extend at least 2 inches below the sidearm of the flask. Locate the
collection flask inside the biosafety cabinet instead of on the floor so the liquid level can be seen
easily, and the flask emptied before it overflows. The second flask (overflow) may be located
outside the cabinet.
B10.4.1.1(a)
If flasks are used at floor level, place them in a sturdy leak-proof container to prevent
breakage and spills. In BSL2 or BSL3 laboratories, the use of Nalgene flasks is strongly
recommended to reduce the risk of breakage associated with infectious materials.
B10.4.2
Vacuum Line Filter
A hydrophobic filter will prevent fluid and aerosol contamination of central vacuum systems or
vacuum pumps. The filter will also prevent microorganisms from being exhausted by a vacuum
pump into the environment. Hydrophobic filters or Vacushields™ are available from several
scientific supply companies (e.g., EMD Millipore Millex™ Filters: Inlet and Outlet for Latex Tubing
or Vacushield™ Vent Device, Pall® Life Sciences).
B10.4.2.1
When working with agents that require Containment Levels 2 & 3, a hydrophobic in-line
vacuum filter shall be used.
B10.4.2.2
Vacuum line filters are disposed of as biological waste and require treatment prior to
disposal.

B11 DECONTAMINATION AND DECOMMISSIONING OF LABORATORY EQUIPMENT

B11.1

General Information
This is not an all-inclusive list of hazards and equipment. It is the responsibility of the PI to ensure
that all equipment is properly and adequately decontaminated. Check with the equipment
manufacturer for recommendations regarding decontamination procedures.
B11.1.1
Regular decontamination of equipment is required in any biological laboratory.
Decontamination logs are required at Containment Level 2 or greater.
B11.1.2
Equipment in need of repairs, maintenance, or removal from areas where rNA/sNA and/or
potentially infectious materials are stored and/or manipulated must be autoclaved or
thoroughly chemically decontaminated then cleared by EHS prior to repairs, maintenance
or removal.
B11.1.3
The Equipment Decontamination Form is to be filled out and submitted to EHS if the
equipment is to be sent for repairs/maintenance or otherwise removed from the laboratory.
This form is available on the EHS website.
B11.1.4
It is the responsibility of the PI to contact the manufacturer to address additional
considerations. Should further questions regarding decontamination exist, please contact EHS.
B11.1.5
Decontaminate all interior and exterior surfaces of the equipment – do not neglect tubing
and other accessories or components.
B11.1.6
Personnel shall always wear appropriate PPE while performing decontamination; at
minimum, gloves, lab coat, and protective eye wear should be worn.

B11.2 Hazard Assessment

Additional hazards may be associated with the equipment (i.e., chemical and/or radioactive). If
other hazards are present, contact EHS.
B11.2.1
Biological Hazard
Disinfect all surfaces with an appropriate chemical agent, or if possible, autoclave parts or
equipment that can withstand autoclave conditions using a biowaste setting (30 minutes @ 121°C,
15 psi). Contact EHS if other hazards are present.

B11.3 EHS Clearance of Decontaminated Equipment

B11.3.1
It is the laboratory’s responsibility to ensure that equipment is thoroughly decontaminated.
Once the equipment has been decontaminated, the PI or lab supervisor will need to fill out and
submit the Equipment Decontamination Form. Upon receipt of the form, EHS will come to inspect
the equipment.
B11.3.2
Equipment shall not be removed from the laboratory for repairs, surplus, disposal, etc. until
EHS has cleared the equipment.

B11.4 Considerations for Common Equipment

This is a guideline – it is not an exhaustive list of equipment, nor may it cover all features of the
equipment listed.
Biological contamination is addressed here. Autoclave items when possible and allow allotted
contact time for disinfectant used during the decontamination process when chemical disinfection
is used. If an item was used for chemical or radioactive storage and/or manipulation, refer to the
CHP or Radiation Safety Manuals as applicable to the equipment and hazards.
B11.4.1
Refrigerators and Freezers
Remove all contents and either appropriately store or dispose of them. Defrost the equipment if
necessary. All liquid should be collected and treated as biohazardous waste. Decontaminate all
surfaces with appropriate chemical disinfectant.
B11.4.2
Incubators
Follow manufacturer’s guidance. Remove thermometers; properly secure any mercury
thermometers. Drain and collect water from water-jacketed incubators for disinfection. If water
doesn’t have a disinfectant present already, disinfect water by autoclaving or chemically by adding
enough bleach to achieve a 10% solution; allow bleach solution to sit for at least 20 minutes then
pour down a drain. If water was visibly contaminated, fill the water jacket with disinfectant. After
the allotted contact time, drain and flush with copious amounts of DI water.
Some incubators have a sterilization cycle. Disinfect all interior surfaces based on the risk
assessment then run the sterilization cycle. Follow with 70% ethanol if a different disinfectant was
used to remove any residue. Some disinfectants can leave residues that can be absorbed by
media and be cytotoxic.
For incubators without a sterilization cycle, autoclave the removable parts able to withstand
autoclave conditions using biowaste settings unless otherwise specified by the manufacturer.
Chemically decontaminate the remaining surfaces.
B11.4.3
Biosafety Cabinets
Biosafety cabinets require decontamination by a third party before the cabinet is removed from a
lab or repaired (B10.1.4.3(b)). Decontamination requires fumigation with formaldehyde, chlorine
dioxide or vapor phase hydrogen peroxide. The company contracted shall provide a certificate of
decontamination once completed. The BSC will require recertification prior to use.
B11.4.4
Laminar Flow Hood
Laminar flow hoods should not need fumigation to disinfect as only non-hazardous items should
have been handled in them and the HEPA filter only filters lab air. Decontaminate all surfaces with
70% ethanol or other desired disinfectant.
B11.4.5
Centrifuges
Remove tube adaptors and the rotor if possible. Autoclave rotors when indicated by manufacturer;
otherwise, soak snap-on lids, adaptors, and rotors in a manufacturer-approved disinfectant
appropriate for agent(s) for appropriate contact time. Rinse with DI/RO water, dry, and follow with
70% ethanol or isopropanol. Disinfect the exterior of the centrifuge in the same manner.
B11.4.6
Water baths
Disinfect water chemically by adding enough bleach to achieve a 10% solution. Scrub inside of
water bath to release any biofilms/mold/algae and allow to sit for 20 minutes. Dispose of water in
drain and chemically disinfect the lid and outside of the bath. Rinse to remove bleach.
B11.4.7
Balances/Scales
Remove parts that can be removed. Disinfect with appropriate disinfectant, follow with 70%
ethanol or water if needed.
B11.4.8
Automated Liquid Handling systems
These systems should have decontamination instructions from the manufacturer. In the absence
of instructions, drain and capture any liquids from the system and autoclave or chemically disinfect
collected liquid by adding enough bleach to achieve a 10% solution, allow contact time and
discard.
Flush system with a 10% bleach solution; follow with a copious amount of DI or RO water. After
the water rinse, flush the system with 70% ethanol (trap waste for EHS disposal) and flush system
with sterile DI or RO water.
B11.4.9
General Reusable Laboratory Supplies
Items such as pipettes, serological pipetting devices, hot plates, stir plates, vortexes, chairs,
furniture, storage cabinets, glassware, etc. should be sanitized with an appropriate disinfectant to
neutralize any agents to which these items have been exposed. Check with the manufacturer
regarding the potential for autoclaving an item. Disassemble equipment where possible, allow
adequate contact time for disinfectant to work and be sure to clean corners, crevices, and crannies
that are hard to reach.

B12 DECOMMISSIONING A LABORATORY SPACE

B12.1 General Information

This requirement is designed to assist PIs who are vacating a laboratory space at the University for
any reason. These guidelines ensure that the laboratory space will be cleared of hazardous
materials so as to protect contractors and other personnel and to expedite laboratory assignment
to new occupants. Also see LSM A25.
The PI is to notify EHS that the laboratory will be vacated 30 days prior to departure or when
known. Upon notification, EHS will inspect the laboratory to identify the hazards that must be
addressed before the PI departs. The laboratory will be inspected again prior to PI departure to
ensure that all hazards are addressed and to verify the clearance of the laboratory space.
If a PI departs before the laboratory is decommissioned, the department assumes the
responsibility of the laboratory and its contents unless otherwise indicated below.

B12.2 Decontamination

The entire laboratory and its contents shall be decontaminated or neutralized prior to the PI
departing the laboratory.
Certain equipment may require decontamination by a third party. Appropriate disinfectants must
be used to address the agent(s) that have been stored and/or manipulated in the laboratory.
Allow adequate contact time for disinfectants and autoclave when possible.
Refer to sections B8 and B11 for more information on disinfectants and decommissioning of
laboratory equipment, respectively.
B12.2.1
All laboratory benches, equipment, glassware, storage areas, shelves, fixtures, and
furniture must be decontaminated with an appropriate disinfectant for agents present in
laboratory.
B12.2.2
Leave hazard labels in place on equipment.
B12.2.3
Fume hoods, glove boxes, and BSCs must be decontaminated. BSCs require a third party for
decontamination if they are to be removed from the work area.

B12.3 Packing and Moving

B12.3.1
Laboratory personnel are responsible for moving the laboratory. Consult the Lab Move
guidelines on EHS’s webpage for guidance.
B12.3.2
Assume all chemicals, biological materials, and gases are regulated. Contact EHS with
questions regarding the transportation of any hazardous materials.
B12.3.3
A PI may transfer biological materials to an existing PI; however, the PI receiving the
materials is responsible for attaining an IBC protocol and containment level for the received
materials when required. See section B4.2.9 for project types that require IBC review and
receive approval before a transfer is made. Contact EHS if you have any questions at 806-742-
3876.
B12.3.3.1
Any biological materials that require an IBC protocol that are not transferred to a new PI
and covered with a protocol become the property of EHS and will be destroyed.

B12.4 Waste Disposal

All chemical, biological, and radioactive waste must be disposed of in their respective waste
streams. See A25.1.1.2.
B12.4.1
Biological Waste
All biological materials must be appropriately destroyed, transferred to a new PI or
moved/shipped to their new location in a DOT/IATA-compliant manner. Contact EHS
regarding the transportation of biological material.
B12.4.1.1
Prior to the PI departing the laboratory, all sharps and other biohazardous waste shall be
disposed of in accordance with Section 8 of the Biosafety Manual.
B12.4.2
General Waste
Any waste (boxes, trash, broken glass, etc.) shall be properly disposed of prior to the PI
departing the laboratory.

B13 TRANSPORT AND SHIPMENT OF BIOLOGICAL MATERIALS

B13.1 Transport of Biological Materials on or to Texas Tech University Property

The following procedures are to be followed when transporting biological materials to or
between laboratories on Texas Tech properties.
Please call EHS at 806-742-3876 if you have any questions regarding the transport of materials on
or to TTU property. The use of personal vehicles is discouraged and done at the owner’s risk.
University vehicles are encouraged for all transport, and as stated in LSM A20.1.5, transport of
DOT-regulated materials on public roadways requires the use of a University vehicle.
B13.1.1
Conditions
B13.1.1.1
Do not take biological materials to non-lab areas or leave items unattended during
transport.
B13.1.1.2
The destination lab must be the same containment level and classification/level as the
materials being transported.
B13.1.1.3
Materials must be properly packaged as outlined in B13.1.2.
B13.1.1.4
The package is not to be opened in transport between laboratories for any reason.
B13.1.1.5
If traveling, have a biological spill kit with you in case of a spill. In the event of a spill,
submit a SCAN. If a spill occurs between laboratories on campus, notify EHS immediately
at 806-742-3876.
B13.1.2
Packaging
B13.1.2.1
For RG1 or materials that require Containment Level 1 biological materials or specimens
that may potentially contain such materials:
(a) Primary specimen container shall be leak-proof, sealed, and labeled with the following
information: Name or initials, date, identifying number, name, or other information.
(b) Primary specimen containers shall be wrapped in absorbent material (if liquid in nature) and
placed in a rigid secondary, leak-proof container with a locking lid.
B13.1.2.2
For RG2 or materials that require Containment Level 2 biological materials or specimens
that may potentially contain such materials:
(a) Primary specimen container shall be leak-proof, sealed, and labeled with the following
information: Name or initials, date, identifying number, name, or other information
(b) Primary specimen container shall be wrapped in absorbent material (if liquid in nature) and
placed in a secondary sealed, leak-proof container (e.g., Ziploc bag).
(c) The packaged material should then be placed in a rigid transport container, such as a
cooler, labeled with biohazard stickers and the name of the PI.

B13.2 General Shipping Information

Shipping and receiving of infectious agents, biological products/specimens, clinical specimens, and
other potentially hazardous substances is controlled by multiple agencies. Regulations are not
always uniform, and permits are often required. These regulations are continually modified, and
new ones are added across regulating entities.
Non-compliance with regulations can result in financial penalties to the shipper and potentially to
the University. Even with training, EHS recommends that the shipper check with applicable
regulatory agencies prior to shipping potentially hazardous materials. See LSM A20.
B13.2.1
Shippers or employees handling hazardous goods (chemical or biological) are required to
take Hazardous Shipper Training pertinent to their duties.
B13.2.1.1
The training must be renewed every two years so long as duties regarding shipping are
ongoing.
B13.2.2
The Department of Environmental Health & Safety is to be notified of all shipments of
biological materials initiated by TTU personnel 2 business days before the shipment is to
occur.
B13.2.3
A copy of all shipping documents must be submitted to EHS for regulated materials before
the shipment departs.
Please contact EHS at 806-742-3876 to schedule your training prior to your shipment. Once
training is complete the certificate is current for 2 years.

B13.3 Permits and Documentation

When applicable, the responsible party (i.e., PI or lab supervisor) must secure permits for
transport prior to shipment and the permit(s) must accompany the shipment and/or
transport.
Permits take time to process. Be proactive in obtaining proper permit(s) for materials you plan on
shipping or receiving well in advance (several months). While it may not take that long to obtain a
permit, allowing ample time will ensure materials can be transported on the desired schedule.
Items that do not require a permit to ship and/or receive may still require special packaging.
B13.3.1
CDC Import Permits
The Centers for Disease Control and Prevention’s Import Permit Program (IPP) regulates the
importation of infectious biological agents, infectious substances, and vectors of human disease
into the United States. Inter-state transfer of previously imported materials also requires the
receiving party to obtain a permit.
Prior to issuing an import permit, IPP reviews all applications to ensure that entities have
appropriate safety measures in place for working safely with the imported materials; anticipate an
inspection by the CDC if you obtain an import permit. More information regarding CDC permits can
be found at: https://www.cdc.gov/cpr/ipp/index.htm.
B13.3.1.1
The CDC Import Permit Program (IPP) sites 42 CFR 71.54 to address the import regulations for
infectious biological agents, substances, and vectors. See their website for the most current
information: https://www.cdc.gov/cpr/ipp/regulations.htm. They also have a decision tool available
to help you determine if an import permit: https://www.cdc.gov/cpr/ipp/etool.htm, as well as, an
FAQ page: https://www.cdc.gov/cpr/ipp/faq.htm
B13.3.2
USDA permits
APHIS or APHIS-PPQ issues permits for export and import for, intra-/inter-state transit of, and
release within the United States of regulated animals/animal products, veterinary biologics,
plants/plant products, pests, organisms, soil, and genetically engineered organisms. See the
APHIS website for more information: https://www.aphis.usda.gov/aphis/resources/permits.
Animal materials (especially food animal-related materials) whether offered for import, export, or
interstate transport, require evaluation through the USDA-APHIS Veterinary Services Permitting
Assistant. Should your animal materials not be subject to a permit, in most cases, a Letter of
Conditions will be automatically generated for the materials. This letter must accompany the
shipment. If a permit applies, you will be routed to the permitting application. There is a "Guide
Me" function for assistance with the permitting assistant or you can reach out to EHS by phone
(806.742.3876) or email ehs.lab.safety@ttu.edu.
B13.3.2.1
Animal and animal products - Includes live animals, semen, embryos, and materials derived from
animals or exposed to animal-source materials such as animal tissues, blood, cells or cell lines of
livestock or poultry origin, RNA/DNA extracts, hormones, enzymes, microorganisms including
bacteria, viruses, protozoa, and fungi. In addition, animal materials including dairy products (except
butter and cheese), and meat products (e.g., meat pies, prepared foods) from countries with
livestock diseases exotic to the U.S.
B13.3.2.2
Veterinary Biologics - Includes vaccines, bacterins, antisera, diagnostic kits, and other products of
biological origin.
B13.3.2.3
Biotechnology products - Includes genetically engineered organisms considered to be regulated
articles.
B13.3.2.4
Plants, Organisms, and Soil
Organism and Soil Permits: Include plant pests such as arthropods and mollusks (insects and
snails); plant pathogens such as fungi, bacteria, nematodes, mycoplasma, viroids and viruses;
biological control agents, bees, Plant Pest Diagnostic Laboratories, Soil Microbe Isolation
Laboratories, Federal Noxious Weeds and Parasitic Plants.
Plants and Plant Products Permits: Include Plants for Planting such as nursery stock, small lots of
seed, and Postentry; Plant Products such as fruits and vegetables, timber, cotton and cut flowers;
Protected Plants and Plant Products such as orchids and Threatened and Endangered plant
species; Transit Permits to ship regulated articles into, through, and out of the U.S.; and Controlled
Import Permits to import prohibited plant materials for research.
Transit Permits: Required by the Animal and Plant Health Inspection Services (APHIS) in advance
of arrival for the unloading, landing or other movement of plants, plant products, plant pests, or soil
in cargo through the United States.
Permit Website: https://www.aphis.usda.gov/aphis/ourfocus/planthealth/import-information/permits
B13.3.3
FWS Permits
U.S. Fish and Wildlife Service permits are required for certain live animals, including bats. Please
call 1-800-344-WILD or go to https://www.fws.gov/permits/?ref=topbar for further information.
Check with the individual states’ wildlife service about permits as well.
B13.3.4
Select Agent Transport
Importation of select agents or biological toxins requires the intended recipient to be registered with
the Select Agent Program and submit required paperwork to obtain approval to import the select
agent or toxin prior to each importation event (see 42 CFR 73 and/or 9 CFR 121). Select agents
are listed online: https://www.selectagents.gov/SelectAgentsandToxins.html. Please contact EHS
at 806-742-3876 if your work includes any of the agents listed.
B13.3.5
Export Control
If you plan to export biological materials, contact EHS for guidance.
The export of a wide variety of microorganisms and toxins of human, plant, and animal diseases
may require a license from the Department of Commerce, Bureau of Industry and Security.
Further information may be obtained by calling the Department of Commerce, Bureau of Industry
and Security at 202-482-4811 or through the internet at https://www.bis.doc.gov/Licensing.
Other agencies such as the FDA or DEA may regulate the goods you wish to transport. Please
consult the FDA and DEA regulations for guidance related to the item you wish to export and do
not rely solely on the Export Administration Regulations for information about other agencies’
export control requirements.
B13.3.5.1
All products and technology that leave the country are subject to TTU OP74.10. Before you travel
or ship items outside the country, consult the TTU Export Control webpage:
https://www.depts.ttu.edu/research/integrity/export-control/ or contact Export Control directly with
questions by email to Allie Matviko, amatviko@ttu.edu.
B13.3.6
Additional Documentation
The shipper is required to initiate and secure all applicable non-permit documentation prior
to transport of the materials. Letters of disclosure, material transfer agreements, SDS sheets,
packing lists, customs declaration forms, hazard declaration forms and other documentation may
be required to successfully complete your shipment.
Additional documentation is often needed in the transfer of scientific materials to and from the
University. Contact EHS at 806-742-3876 if you have questions regarding the documentation
needed for your shipment.
A Material Transfer Agreement (MTA) is required if the biological material was developed or
recovered at Texas Tech University/Texas Tech University Health Sciences Center as it is
considered the proprietary property of Texas Tech University/Texas Tech University Health
Science Center. The MTA form is available on the Office of Research Services website at:
http://www.depts.ttu.edu/research/ors/preaward/forms-boilerplates.php. Questions regarding the
need for an MTA can be addressed by Cui Romo: cui.romo@ttu.edu.

B13.4 Packaging

Items are required to be properly identified, packaged, marked, and labeled for shipment.
Various agencies such as the International Air Transport Association (IATA) and the Department of
Transportation (DOT) have developed guidelines and procedures to facilitate the safe shipment of
potentially infectious substances and other hazardous materials.
As previously mentioned, these regulations are frequently updated. It is important to check with
the carrier you have chosen (and country of destination if shipping internationally) to determine
their specific requirements for shipping the material(s) in question.
B13.4.1
Permitting is not associated with the need for, or lack thereof, special packaging and labeling
requirements. Call EHS at 806-742-3876 if you have questions about a shipment and/or to
schedule your training prior to your shipment.
Reuse of packaging is discouraged; consult EHS prior to re-use of packaging.
B13.4.2
Shippers are responsible for procuring appropriate packaging for their shipment