Principles and Practices of Biosafety - San Diego State University

Principles and Practices of 

Biosafety 

Environmental Health and Safety 

San Diego State University

Abbreviations 

• BSC – Biosafety Cabinet 

• BSL – Biosafety Level 

• ABSL – Animal Biosafety B 

Level 

• BSO – Biosafety Officer 

• BUA – Biological Use Authorization 

• CA – California 

• CDC – Center for Disease Control and 

Prevention 

• DGR – Dangerous Goods Regulations 

• DOT – Department of Transportation 

• EHS – Environmental Health and 

Safety 

• EPA – Environmental Protection 

Agency 

• HMR – Hazardous Materials 

Regulations 

• IACUC – Institutional Animal Care 

and Use Committee 

• IATA – International Air Transport 

Association 

• IBC – Institutional Biosafety 

Committee 

• IRB – Institutional Review Board 

• NIH – National Institute of Health 

• OSHA – Occupational Health and 

Safety Administration 

• PI – Principal Investigator 

• PP – Physical Plant 

• PPE – Personal Protective Equipment 

• PS – Pubic Safety 

• SD – San Diego 

• USPS – U.S. Postal Service

Introduction 

The management of biological hazards through the 

proper application of engineered containment and 

administrative controls is referred to as biosafety 

or biohazard control. 

Biosafety or biohazard control is a team effort 

involving the PI, research lab personnel, BSO, 

IBC, IRB, IACUC, EHS, PP and PS.

Regulatory Requirements and 

Guidelines 

• NIH Guidelines for Research Involving 

Recombinant DNA Molecules (April 2002) 

• CDC/NIH Biosafety in Microbiological and 

Biomedical Laboratories (May 1999) 

• CAL/OSHA Bloodborne Pathogen Standard 

• CA Medical Waste Management Act 

• DOT Transportation of Hazardous 

Materials

SDSU Biosafety Requirements 

and Guidelines 

• Policies established by the Institutional Biosafety 

Committee (IBC) that meet or exceed applicable 

guidelines and regulations for use of RG 2 and 

RG 3 biohazardous materials or agents as well as 

non-exempt NIH Recombinant DNA research. 

• Policies established by Environmental Health and 

Safety (EHS) that meet or exceed applicable 

regulations and guidelines for minimizing 

bloodborne pathogen exposure and disposal of 

biohazardous wastes.

Biohazardous Material 

• Infectious microorganisms 

(bacteria, viruses, fungi, 

parasites, prions, 

rickettsiae, etc.) affecting 

humans and animals 

• Diagnostic (clinical) 

specimens 

• Recombinant DNA (viral 

vectors, gene therapy, 

cloning) 

• Genetically Modified 

Microorganisms 

(transgenic plants and 

animals) 

• Human and non-human 

primate cells, cell culture 

(primary and 

immortalized), tissues, 

blood (whole blood or any 

components) and body 

fluids 

• Animal or plant cells, cell 

cultures, fluids, tissues or 

derived wastes which 

may contain pathogens 

• Animals known to be 

reservoirs of zoonotic 

disease

Institutional Biosafety Committee 

and 

Biological Use Authorization

Biological Use Authorization 

(BUA) Application 

• BSL 1, ABSL 1 and NIH-Exempt 

Reviewed and approved by BSO 

• BSL 2 or 3, ABSL 2 or 3 and NIH- Non-exempt 

Reviewed and approved by IBC 

Currently, no facilities at SDSU meet the 

minimum criteria for BSL 4; thus this type of 

work is prohibited.

Submission of New BUA 

• New infectious agents 

• New cell lines 

• New vector system 

• Enhanced replication or infectivity 

• Expression of toxic products 

• Partial genomes increased to more than two-thirds 

thirds 

of whole genome 

• New or altered procedures that pose increased risk 

(e.g., aerosol or other type of exposure) 

• Work with non-human systems being changed 

into work with human system

SDSU Biosafety Approval 

Institutional Biosafety Committee (IBC) 

Protocol Review 

Biological Use Authorization (BUA) with 

respect to containment level (biosafety 

level) 

Training 

Inspection

Principal Investigator 

• Develops specific protocols to ensure the safe use 

of biohazardous materials. 

• Submits a BUA application and obtains approval 

from IBC prior to commencement of work. 

• Complies with specific biosafety protocols, 

practices and procedures described in the 

biohazard control manual. 

• Ensures that all laboratory staff are appropriately 

trained on biosafety. 

• Reports any significant problems, violations of 

the policies, practices and procedures, or any 

significant research-related related accidents and/or 

laboratory acquired infection to the BSO 

immediately.

Laboratory Staff 

• Comply with the specific biosafety 

protocols, practices and procedures 

described in the biohazard control manual. 

• Report to the PI or the lab manager all 

problems, spills or violations in procedure 

immediately.

Risk Group 

and 

Biosafety Level

Classification of Infective Agents 

by Risk Group 

• Pathogenicity 

• Infectious Dose 

• Mode of Transmission 

• Host Range 

• Availability of Effective Preventive 

Measures and Treatment

Classification of Infective Agents 

by Risk Group 

Risk Group 1 Risk Group 2 Risk Group 3 Risk Group 4 

Severity of 

Disease 

unlikely to 

cause human 

or animal 

disease 

can cause disease, 

unlikely to be 

serious, effective 

treatment and 

preventive measures 

available 

can cause serious 

disease, does not 

ordinarily spread 

from one person to 

another, effective 

treatment and 

preventive measures 

usually available, 

exposure route: 

inhalation (often) 

likely to cause serious or 

lethal disease, can be 

readily transmitted from 

one individual to another, 

effective treatment and 

preventive measures are 

not usually available, 

transmission: direct, 

indirect, inhalation 

Host Range 

human 

(healthy adult) 

and animals 

human (healthy 

adult) and animals 

human (healthy 

adult) and animals 

human (healthy adult) and 

animals 

Individual 

Risk 

low 

moderate (potential 

hazard) 

high 

high 

Community 

Risk 

low low low high

Classification of Containment by 

Biosafety Levels 

• Practices and Procedures 

• Containment Equipment (Primary Barriers) 

• Containment Facility (Secondary Barriers) 

Note: CDC/NIH has 4 biosafety level classifications 

currently in use. Each level is appropriate for: 

Operations Performed 

Routes of Transmission (ingestion, inoculation, 

inhalation, mucous membrane exposure) 

Laboratory Function

Risk Group and Biosafety Level 

Classifications 

• Not appropriate to use risk group or 

biosafety level when assessing toxins. 

• Can be found in the American Biological 

Safety Association website: 

www.absa.org/resriskgroup 

• Risk Groups and Biosafety Levels are not 

always the same!

Bloodborne Pathogen 

Standard

Introduction 

• California Code of Regulations, Title 8, Sec 5193 

• Applies to all employees who could “reasonably 

anticipate” as a result of performing their job tasks 

contact with blood and other potentially infectious 

material (OPIM) i.e., body fluids, unfixed tissues 

or organs 

• Limit occupational exposure to blood and OPIM 

which could result in transmission of bloodborne 

pathogens i.e., Hepatitis B virus (HBV), HIV 

• Requires a written Exposure Control Plan

Elements of an Exposure Control 

Plan 

• Exposure Determination 

• Method of Compliance 

• HIV, HBV and HCV Research Laboratories 

• Hepatitis B Vaccination and Post-Exposure 

Evaluation and Follow-Up 

• Hazard Communication 

• Record Keeping 

• Evaluation of Exposure Incidents 

• Sharps Injury Log

Exposure Determination 

• Exposure evaluation based upon the job 

description. 

• Exposure evaluation based upon reasonably 

anticipated contact (skin, eye, mucous 

membrane, parenteral contact, etc.) with 

blood or other potentially infectious 

materials resulting from performing the 

assigned tasks.

Method of Compliance 

• Universal Precaution – treating all human blood and 

certain human body fluids as if infectious for bloodborne 

pathogens 

• Engineering and Work Practice Controls 

 

 

 

 

 

Needleless system or needles/sharps with engineered sharps injury 

protection 

Needles and other sharps shall not be recapped, bent or broken 

Needles and other sharps shall be disposed in rigid, puncture- 

proof, leak resistant and properly labeled sharps container 

Sharps container shall be closed immediately prior to removal or 

replacement to prevent spillage or protrusion of contents during 

handling or transport 

Specimens of blood or OPIM shall be placed in a closable, 

leakproof, properly labeled red bag prior to handling, collection n or 

transport 

• Personal Protective Clothing and Equipment

Hepatitis B Vaccination and 

Post-Exposure Follow-Ups 

• Hepatitis B vaccination shall be provided at 

no cost to employee who has a potential for 

becoming exposed to blood or OPIM. 

• Post-exposure follow-ups shall be provided 

if an occupational exposure occurs.

Hazard Communication 

1. Signs and Labels 

• Signs shall be posted at the entrance to the work areas 

which shall bear: 

Name of infectious agent 

International symbol for biohazard in fluorescent 

orange-red 

red 

Special requirements for entering the area 

Name and telephone number of lab director or other 

responsible person 

Warning labels shall be affixed to containers of 

infectious wastes, refrigerators and freezers 

containing blood or OPIM, or other containers used 

to store or transport blood or OPIM. 

• Labels shall have the international symbol for biohazard 

in fluorescent orange-red 

red 

2. Training

Pathogenic Microbiology

Bacterial Laboratory Acquired 

Infections 

• 76% of exposures occurred in clinical labs; 

6% in vaccine manufacturing facilities; 8% 

in research labs. 

• Exposure modes: 60% inhalation, other 

exposure modes included ingestion 

(intentional, poor technique such as mouth 

pipetting, smoking and eating in the lab) 

and secondary transmission.

Viral Laboratory Acquired 

Infections 

• >70% associated with research labs; 32% of all 

viral LAIs associated with animals 

• 18% of total were Hantavirus; of these, 8% were 

working with known infectious material or rodents 

- others thought they were working with 

uninfected rodents 

• 16% were in clinical labs; rest were in production 

or field work 

• Major exposure modes: inhalation, percutaneous 

(especially from animals)

Rickettsial Laboratory Acquired 

Infections 

• All rickettsial LAIs were associated with research 

laboratories 

• 95% of overt infections were by Coxiella burnetii; 

remainder were Murine typhus 

• All Q fever cases by inhalation; all infected staff 

worked with or were in close proximity to sheep 

• Remaining cases were by percutaneous, 

inhalation, mucous membrane or unknown 

exposure modes

Molecular Biology

It’s a Matter of Perspective 

• The investigators who submit IBC protocols 

want to perform their experiments safely. 

• However, their perception of the risks 

involved will not necessarily be the same as 

that of a biosafety professional.

Risk Assessment 

The following risk assessment will identify the 

biological containment system to be used: 

Properties of the donor organism 

Nature of the DNA sequences that will be 

transferred 

Properties of the recipient organism 

Properties of the environment

Biological Expression System 

Most routine genetic engineering experiments 

can be performed safely in E. coli 

K12/pUC18 at BSL 1 provided the inserted 

foreign DNA sequences do not require a 

higher BSL.

Donor Organism and Cloned DNA 

Insertion of well-characterized DNA sequences that 

are unlikely to be involved in pathogenicity may 

not require additional safety measures. 

In cases where these sequences are not characterized, 

a situation that is typically encountered when a 

library of genomic DNA of an organism is being 

established, a higher BSL will be required. 

Cloning of genes coding for proteins that have 

potential pharmacological activity such as toxins 

may therefore require higher BSL.

Viral Vectors for Gene Transfer 

Although viral vectors used in gene therapy or 

gene transfer are replication-defective, they 

should be handled at the same BSL as the 

parent viral vector from which they are 

derived since the virus stocks may be 

contaminated with replication-competent 

competent 

viruses, which are generated by rare 

spontaneous recombination events in the 

complementing cell line.

Transgenic and “Knock-Out” 

Animals 

Animals carrying foreign genetic information 

(transgenic animals) should be handled in the 

containment level appropriate to the characteristics 

of the products of the foreign genes. For each new 

line of transgenic animal, the routes by which the 

animals can be infected, the inoculum size 

required for infection, and the extent of the virus 

shedding by the infected animal must be 

determined. 

Animals with targeted deletions of specific genes 

(“knock-out” animals) do not generally present 

particular biological hazards.

Human and Other Primate 

Cells and Tissues

Human Source Material 

• Blood and blood products 

• Vaginal secretions 

• Semen 

• Amniotic fluid 

• Unfixed tissues 

• Cerebrospinal, synovial, 

pleural, pericardial and 

peritoneal fluids 

• Cell cultures 

• Saliva 

• Urine 

• Tears 

• Sputum 

• Feces 

• Vomit 

• Other excretions and 

secretions 

Second column not covered in Bloodborne Pathogen Standard, possibly not 

occupationally related.

Human Source Material 

• May transmit infectious agents 

• Imperfect knowledge of infectious status 

Incubation period (asymptomatic) 

No test for every pathogen 

• Most tissues and body fluids 

Bloodborne Pathogens (HBV, HCV, HIV, 

HTLV-1) 

Pathogens causing Malaria, Syphilis, 

Babesiosis, Brucellosis, Leptospirosis, 

Arboviral infections, Relapsing fever, 

Creutzfeldt-Jacob disease and viral 

hemorrhagic fever

Cell Culture Risks 

• Contaminating pathogenic agents 

natural (often zoonotic) or inadvertent 

~20 LAIs from primary cultures in last 30 years 

e.g., Herpes B (CHV-1), prions 

• Oncogenic potential 

could be oncogene or oncogenic agent 

e.g., HPV-18, MPMV genomes in HeLa cells 

• Unexpected (adventitious) agents 

e.g., SIV, STLV, SV5 in primate cells, HHV-8 

in BCBL-1 1 cells 

• Hazardous chemicals added to culture medium

Cell Culture under Bloodborne 

Pathogen Standard 

• ABSA requested OSHA’s s interpretation in 1994: 

Do human cell cultures fall under the 

Bloodborne Pathogen (BBP) Standard 

• Response: 

All primary human cell cultures (explants) and 

subsequent in vitro passages fall under the 

BBP Standard 

To be exempted from the BBP requirements, 

cell strains and lines must undergo testing and 

characterization (documented) for bloodborne 

pathogens (not just HBV, HCV and HIV)

Cell Culture Safety 

• Extend Universal/Standard Precautions to all 

human and animal cell cultures 

Consider working at BSL 2 (most work 

there already to protect the cell cultures) 

Handle all cultures in a biosafety cabinet 

If human origin and not demonstrated to 

be free of human bloodborne pathogens, 

adhere to requirements of the BBP Standard 

Wear PPE appropriate to human source 

material

Summary 

Human Source Materials 

• May be regulated 

• Can be biohazardous 

• Use Universal Precautions at 

all times 

• Visible blood means 

increased risk 

• Don’t t consider “normal” 

source 

Human and Non- 

human Primate Cell 

Cultures 

• Treat human cultures as 

possible biohazards 

• Beware of non-human 

primate cells 

• Beware of CNS, corneal, 

pituitary cells 

• Some cells may be OK at 

BSL 1

Animals and Allergens

Risk Assessment for Work with 

Research Animals 

• Risks associated with the research agent used in 

the animal 

chemical, physical, biological 

• Risks associated with the species of animal used 

zoonotic agents 

• Risks associated with animal maintenance 

ergonomic factors, bites, scratches, allergens

Risks Associated with the Agent 

Used 

• Chemical agents 

carcinogens, mutagens 

toxic chemicals 

anesthetics 

• Physical agents 

radiation 

heat 

sound

Risks Associated with the Agent 

Used 

• Potentially biohazardous agents 

deliberate use of an infectious agent in 

animals for research purposes 

maintenance of infected animal for 

duration of experiment 

sacrifice, necropsy and harvesting of 

agent or infected tissue

Transmission of Biohazards 

During Work with Animals 

Airborne 

• Release of infectious aerosols by animal by 

sneezing, coughing 

• Release during nasal infection or aerosol 

challenge 

• Aerosolization from bedding and excreta 

• During surgical procedures 

• During birthing



Direct Inoculation 

• Needlesticks during injection/inoculation 

process 

• Bites and scratches from infected animal



Direct exposure of mucous membranes 

(by splash or splatter) 

• During surgical procedures 

• During injection 

• During necropsy



Indirect transmission and ingestion 

• From contaminated hands or gloves to mouth 

• Facial contamination directly from animal 

• Transfer of parasites by animal handling 

Indirect transmission with eye or mucous 

membrane exposure 

• Dust from bedding 

• Splash during cage washing 

• “Dirty” environment

Risk Reduction: Containment of 

Infectious Agent 

• Containment must include: 

Primary containment 

Enclosed filtered caging system 

Biosafety cabinets 

Safety equipment 

PPE 

Secondary containment 

The containment facility 

• Negative pressurization 

• Nonrecirculated air supply 

• Ventilation must consider wellbeing of animal

Containment Caging Systems 

• No Containment 

Open (standard) cage 

• Some Containment 

Filter top cage 

(microisolator cage) 

• Full Containment 

Fully enclosed in 

ventilated rack


• Microisolator Cage 

works like a Petri dish 

open gaps around lid edge allow limited air 

exchange 

may lead to more labor intensive husbandry due 

to moisture and ammonia buildup


• Individual cages sealed into rack with 

supplied air under negative pressure 

• Both supply and exhaust usually HEPA 

filtered 

• Ventilation must control humidity and 

buildup of ammonia


• Can install cages in class III biosafety 

cabinet 

• Cages are completely contained with glove 

port access 

• Very motion-limiting 

• Transfer in and out may be an issue


• BioBubble (Ft. Collins, CO) makes soft- 

wall ventilated enclosures 

• Can be containment or barrier style 

• Large equipment can be surface-mounted in 

wall

Special Animal Housing Situations 

• Barrier colonies 

Special breeds - often immunocompromised, 

“fragile”,, expensive (SCID-Hu, nude athymics) 

Transgenics - often even more fragile and 

expensive (knockouts, microinjected, combos) 

Specific pathogen-free (SPF) - bred and raised 

to be missing certain specific microorganisms 

• Isolation colonies 

Extensive SPFs and defined flora animals 

Gnotobiotes (an entirely different animal!)

Zoonoses 

• Zoonotic disease: : A disease of animals that 

can be transmitted under natural conditions 

and cause disease in humans 

Wild caught animals most hazardous 

Random source animals (e.g., from a 

pound) are also a risk 

Purpose bred animals pose least risk

Some Animals and Their 

Zoonoses 

• Animal 

Macaque monkeys 

Sheep 

White mouse 

Dogs, cats, skunks, 

raccoons, bats 

Cattle, NHP 

Cats 

Parrots, macaws 

Chickens 

• Disease 

Herpes B virus 

Q fever 

Hantavirus 

Rabies 

Tuberculosis 

Toxoplasmosis 

Psittacosis 

Avian influenza

Rodent Zoonoses 

• Rat bite fever (Streptobacillus( 

moniliformis, 

Spirillum minus) 

transmission: direct contact (bites) 

• Lymphocytic choriomeningitis (LCM, a virus) 

transmission: inhalation 

• Leptospirosis (Leptospira( 

spp.) 

transmission: inhalation 

• Others include ringworm (fungal), scabies (mites, 

an ectoparasite)

Transmission of Zoonoses 

• Enteric route (fecal/oral) 

Salmonella, Shigella, Campylobacter, 

Giardia, Toxoplasma, Cryptosporidium, 

Entamoeba, , Hepatitis A 

• Respiratory route 

Q fever, Chlamydia, , Measles 

• Skin contact 

Ringworm (Tinea), Measles, Monkeypox

Control of Zoonoses 

• Get information on 

species and agent 

• Quarantine animals prior 

to use 

• Use Engineering controls 

 

facility construction and 

secondary barriers 

• Consider the need for 

containment caging 

• Use Administrative 

controls 

 

• Use PPE 

 

written SOPs and 

manuals 

additional protection for 

worker 

• Practice good facility and 

personal hygiene 

• Provide staff training

Laboratory Acquired Allergies 

(LAA) 

• Significant occupational disease 

• Affects >30% of all personnel working with 

animals 

• No minimum safe exposure levels to allergens 

have been established 

• Animal allergens found in hair, dander, urine, 

saliva, serum 

fel-d-l l cat allergen (in saliva and thus on skin) 

is one of the strongest allergens known for 

humans

Sources of Exposure to LAA 

• Hair and dander shed 

from animal 

• Urine and feces dried 

in bedding 

• Particulates shed from 

bedding material 

• Animal saliva

Routes of Exposure to LAA 

• Inhalation of airborne allergens 

during cage changing 

during animal handling 

• Skin or eye contact 

usually indirect by touching skin, eyes 

• Percutaneous exposure 

animal bites (saliva)

Risk Factors for Development of 

LAA 

• Exposure to allergens 

duration 

frequency 

intensity 

• Previous allergic conditions 

• Other predisposing conditions 

illness 

Immunocompromised 

pets

LAA: Exposure Control 

• Engineering Controls 

enclosure 

dilution ventilation 

• Administrative Controls 

reduce time with animals 

reduce density of animals 

housekeeping practices 

• Personal Protective Equipment 

respirators and clothing 

• Medical Surveillance

Disinfection

Why Disinfect 

• To reduce or eliminate exposure risk 

Biohazard waste disposal 

Spill cleanup 

Routine surface decontamination 

• To eliminate contamination risk 

Preparation of microbiological media and 

supplies 

Preparation of work area for cleanliness-critical 

critical 

tasks

Resistance to Disinfectants 

• Prions 

• Bacterial spores 

• Coccidia 

(Cryptosporidium) 

• Mycobacterium 

• Nonlipid viruses (Hep A, 

Polio) 

• Fungi 

• Rickettsiae, Chlamydiae 

• Vegetative bacteria 

• Lipid-containing viruses

Classes of Disinfectants 

• Chlorine 

• Iodine 

• Alcohol 

• Phenolics 

• Quaternary Ammonium 

• Glutaraldehyde 

• Hydrogen peroxide

Factors Influencing Efficacy 

• Surface/Topography – uneven, cracked or pitted 

surfaces especially wooden surfaces can hide 

microorganisms and are difficult to disinfect 

• Temperature - elevated temperatures may 

enhance germicidal action but also evaporation 

rate 

• Relative Humidity – many disinfectants have 

optimal relative humidity range for maximum 

effectiveness 

• Water Hardness – some disinfectants may be less 

effective when diluted in hard water

• Organic Load 

Blood, sputum, milk, bedding, feed, 

manure 

Proteins physically protect and stabilize 

many microorganisms 

Adverse effect on action of many 

disinfectants

• Concentration 

In most cases, the higher the 

concentration, the more rapid the kill 

Consider potential damage to surfaces or 

tissues 

Reducing concentration to avoid 

damage will require additional contact 

time 

Ultimately, disinfectant will no longer 

be active enough to be useful

• Contact Time 

Disinfectants should be effective with a 

short contact time 

Manufacturer’s s recommended contact 

time may be unrealistic under in-use 

condition 

Contact time may depend on the method 

of application 

For surface applications, loss by 

evaporation may require frequent 

applications to achieve contact time

Some Other Factors 

• Dirt, grease and oils – all can protect the organism 

and will repel water based disinfectants 

• Types of microbes present – spores, vegetative 

cells, viruses 

• Dried spills (from media, buffers) can protect 

microorganisms from contact with the disinfectant 

• pH 

• Age of the product/solution 

• Method of application (spray vs. wipe) 

• Rate of application 

• Storage condition

Medical Waste Disposal 

(Biohazardous and Sharps Wastes)

Who Regulates Medical Waste 

• Federal 

EPA (40 CFR part 60.51c) 

DOT (49 CFR Part 173.134) 

OSHA (29 CFR Part 1920.1030b) 

USPS (39 CFR 111.1) 

• State CA Health and Safety Code 

• Local SD Code of Regulatory Ordinance

Medical Waste 

…biohazardous waste and/or sharps waste 

that is produced or generated as a result of 

diagnosis, treatment or immunization of 

human beings or animals; research 

pertaining thereto; production or testing of 

biologicals or removal of regulated waste 

from a trauma scene.

Forms of Medical Waste 

• Solid 

Labware (flasks, tubes, plates, bottle, vials) 

Pipettes (could also be sharps) 

Lab waste (stocks, specimens, cultures, swabs, 

vaccines) 

Gloves, apparel, wipes 

• Liquid 

Aspirates, culture fluids, rinses, washes 

Sera, body fluids 

• Sharps 

Anything with a point or edge capable of 

piercing or cutting

Medical Waste Does Not Include: 

• Waste generated in food processing 

• Urine, feces, saliva, sputum, nasal secretions, 

sweat, tears, or vomitus, unless it contains fluid 

blood 

• Medical solid wastes i.e., paper towels or empty 

specimen containers that are not biohazardous, 

bandages/dressings containing dried blood 

• Hazardous waste, radioactive waste, household 

waste 

• Waste generated on agricultural or livestock 

practices on a farm or ranch

Biohazardous Waste 

• Laboratory Wastes 

Human or animal 

specimen cultures 

Cultures and stocks of 

infectious agents 

Wastes from production of 

biologicals, live and 

attenuated vaccines, 

culture dishes and devices 

• Pharmaceuticals 

• Human surgery specimens or 

tissues, including those fixed 

in fixatives 

• Animal parts, tissues, 

fluids, or carcasses 

• Wastes containing 

recognizable fluid blood 

or blood products, 

containers with fluid 

blood, blood from animals 

• Wastes contaminated or 

containing 

chemotherapeutic agents

Sharps Waste 

… means any device having acute rigid corners, 

edges, or protuberances capable of cutting or 

piercing. 

• Needles, needles with syringes, contaminated 

syringes, blades, needles with attached tubing 

• Broken glass items i.e., Pasteur pipettes, blood 

vials contaminated with biohazardous waste

Mixed Waste 

… means mixtures of medical and nonmedical 

waste. Mixed waste is medical waste, except for 

all of the following: 

“Mixed Waste Hierarchy”

Biohazard Bag 

…means a disposable red bag that is 

impervious to moisture and has a strength 

sufficient to preclude ripping, tearing, or 

bursting under normal conditions of usage 

and handling of the waste-filled bag.

Containment and Storage 


• Must be segregated from other types of wastes 

• Must be contained in “biohazard bags” 

Bags must be red. 

Bags must be labeled either with the word 

“Biohazardous” or with the biohazard symbol 

and the word “Biohazard”. 

Bags must also be labeled with the generator’s 

name, address and phone number. 

Bags must be securely tied to prevent leakage 

or expulsion of contents. 

Bags must be placed in a rigid container for 

storage, handling and transport.

• Containers 

Containers shall be leak resistant, have tight-fitting 

covers, kept clean and in good repair. 

Containers may be of any color and shall be labeled 

with the word “Biohazardous” or with the biohazard 

symbol and the word “Biohazard” on the lid and on the 

sides so as to be visible on any lateral direction. 

• Reusable containers shall be washed and decontaminated 

unless protected from contamination by disposable liners 

or bags. 

• Reusable containers shall be washed to remove visible soil 

and decontaminated by: 

Exposure to hot water (180°F) for 15 secs. 

Exposure to the following sanitizer for 3 mins. 

Hypochlorite soln. (500 ppm avail. Chlorine) 

Phenolic soln. (500 ppm active agent) 

Iodoform soln. (100 ppm avail. Iodine) 

Quaternary ammonium soln. (400 ppm active agent)

Sharps Container 

…means a rigid puncture resistant container 

that, when sealed, is leak resistant and 

cannot be reopened without great difficulty.

Containment and Storage 

Sharps Waste 

• Must be segregated from other types of wastes. 

• Must be contained in “sharps container”. 

• Tightly close or tape closed the lid of a full sharps 

container ready for disposal. 

• Store sharps container ready for disposal for not 

more than 7 days. 

• Label sharps container with the word “Sharps 

Waste” or the biohazard symbol and the word 

“Biohazard”. 

• Must also be labeled with the generator’s s name, 

address and phone number that is legible and 

easily visible on the outside of the container.

Disposal 

• Take biohazard bag and sharps container to 

designated Accumulation Sites: 

Life Science, Room 14 

Student Health Service, outside shed 

Generators at other locations may call EHS at 

(619) 594-6778 for biohazard waste pick-ups 

• A biowaste vendor will collect biohazard bag and 

sharps container for disposal on a weekly basis 

from Life Science and Student Health Services. 

• Biohazardous and sharps wastes will be 

autoclaved while animal carcasses will be 

incinerated.

Solid Medical Waste Collection 

Must be rigid, puncture-proof, leak-proof 

Not acceptable in CA 

Labels have to be affixed on all 4 sides of the container.

Sharps Waste Collection 

Sharps containers

What’s Wrong with these 

Pictures 

Left: Sharps sticking out of Sharps Waste container. 

Right: Sharps Waste container past full line. No generator label.


Pictures 

Left: Bottle not labeled. 

Right: Cardboard box is not allowed for liquid waste. No labels. No lid.


Pictures 

Left and Right: Cardboard box is not an appropriate Sharps Waste container. 

No labels. No lids.


Pictures 

Left: Red bag should be inside the secondary container. Cardboard box is not an acceptable 

secondary container. 

Right: Bag must be red. Secondary container does not have to be red. No biohazard label. 

Red bag on floor ready for disposal must be transported to the accumulation site immediately.


Pictures 

Left: Do not fill red bags completely. Replace more often. 

Right: No biohazard label. Red bag on floor ready for disposal must be 

transported to the accumulation site immediately.


Pictures 

Left: Do not deface container. Incorrect label placed on container (need generator label). 

Right: Red bag must be transported in a secure secondary container to the accumulation 

site. Red bag must have biohazard label and generator label.


Pictures 

Left: Proper Sharps Waste container not used. No generator label. 

Right: Generator label should be on the outside of the red bag. Secondary container needs 

biohazard label on all visible sides including top. Use appropriately sized red bag for 

secondary container.


Pictures 

Left: Incorrect label placed on container (need generator label). Keep lid closed when not in 

use. 

Right: No lid. Use appropriately sized red bag for secondary container. Secondary container 

needs biohazard label on all visible sides including top.

Containment Equipment and 

Facilities

Biocontainment 

• The principle of holding or being capable of 

holding or including within a fixed limit or 

area 

• Preventing the unintentional release of 

biological agents through a combination of 

laboratory practices, containment 

equipment (primary barrier) and laboratory 

facility design (secondary barrier)

Primary Barrier 

• Primary barriers contain the agent at the 

source 

• Equipment/Engineering Control 

Biological safety cabinet, fumehood, 

glove box, animal housing, centrifuge, 

fermenter

Secondary Barrier 

• Secondary barrier is the structure 

surrounding the primary barrier 

• Facility/Engineering Control 

Rooms, building 

• Types of Facilities 

Basic laboratory 

Containment laboratory

Primary Barriers - Equipment 

• Personnel Protection 

Any aerosol generated within the cabinet is 

contained and kept away from the researcher 

• Product Protection 

Air within the work space of the cabinet has 

been filtered so that is is virtually free of 

airborne particles and organisms; thus 

protecting the work from outside contamination 

• Environmental Protection 

Aerosols generated within the unit are removed 

from the air before the air is discharged

Ventilation Equipment 

Classes and Types

Chemical Fume Hood 

• 100 fpm face velocity 

• Offer only personnel protection 

• Always exhaust air to the outside 

• Do not offer protection to the product or the environment, 

as there is no filtration of intake and exhaust air 

(Sometimes air cleaning treatment is added to the exhaust.) 

• Do draw contaminants in the laboratory air directly over 

the product being worked on 

• Used for work with chemical hazards

Any Comments 

Fumehood - keep hood clean, sash should be closed when hood is not in 

use, equipment should be 9” from sash

Clean Bench / Laminar Flow Hoods 

• Provide product protection only 

• Product protection is provided by creating a unidirectional 

airflow generated through a HEPA filter 

• Discharge air goes directly into workroom 

• Applications 

– Any application where the product is not hazardous but 

must be kept contaminant free 

– Preparation of non-hazardous intravenous mixtures and 

media 

– Particulate free assembly of sterile equipment and 

electronic devices 

• Eliminate Clean Bench in containment laboratory

Biological Safety Cabinets 

• Designed to contain biological hazards 

• Inward airflow for personnel protection 

• HEPA filtered exhaust air for environmental protection 

• Supply air HEPA filter for product protection (except 

Class I) 

• Separated into Classes and Types 

– Class I 

– Class II 

• Type A1, A2 

• Type B1, B2 

– Class III 

• Microbiological studies, cell cultures, pharmaceutical 

research and procedures…

Class I Cabinet 

• 75 fpm face velocity 

• Provides personnel and environmental protection 

• No product protection 

• Requires an exhaust blower to pull the air through 

- usually to the outdoors 


– Housing centrifuges, fermenters 

– Cage dumping in an animal lab 

– Aerating cultures

Class II Cabinets 

• Ventilated cabinet 

• Provides personnel, product, and environmental 

protection 

• Open front with inward airflow for personnel 

protection 

• Downward HEPA filtered laminar airflow for 

product protection 

• HEPA filtered exhaust air for environmental 

protection

Any Comments 

BSC - remove unnecessary objects, keep grill at front of cabinet unobstructed

Primary Barriers 

Personnel 

Product 

Environment 

Chemical Fumehood 

x 

Laminar Flowhood 

x 

Class I Biosafety Cabinet x x 

Class II Biosafety Cabinet x x x 

Class III Biosafety Cabinet x x x 

Isolators x x x

Types of Biosafety Cabinets 

NSF/ANSI Standard 49 – 2002 

Type 

Face velocity 

(lfpm) 

Airflow Pattern 

Radionuclides/ 

Toxic Chemicals 

Biosafety 

Level(s) 

Product 

Protection 

Class I 75 In at front; rear and top through 

HEPA filter 

No 2, 3 No 

Class II 

Type A1 

75 

70% recirculated through HEPA; 

exhaust through HEPA 

No 2, 3 Yes 

Class II 

Type A2 

100 

30% recirculated through HEPA; 

exhaust via HEPA and hard ducted 

Yes (Low 

levels/volatility) 

2, 3 Yes 

Class II 

Type B1 

100 

No recirculation; total exhaust via 

HEPA and hard ducted 

Yes 2, 3 Yes 

Class II 

Type B2 

100 

Same as B1, but plena under 

negative pressure to room and 

exhaust air is ducted 

Yes 2, 3 Yes 

Class III 

NA 

Supply air inlets and exhaust 

through 2 HEPA filters 

Yes 3, 4 Yes

Biological Safety Cabinet 

Certification 

• First Certification 

• Annually 

• When moved 

• When filter is changed 

• When repaired or modified 

Note: Certification is paid by the researcher, not EHS

Other Primary Barriers- 

Engineering Control 

• Gasketed blenders, homogenizers 

• Cotton plugs, filters for flasks in shakers 

• Filtered pipette tips 

• HEPA and hydrophobic vacuum line filters 

• Plasticware substituted for glassware 

• Gas burners with shield, microincinerator 

• Centrifuges 

Interlock, solid cover, safety buckets, O-ringsO

Secondary Barrier- Facilities 

Laboratory Biosafety Level 2 

• Lockable doors (a must for restricted agents) 

• Sink 

• Bench tops impervious and easily cleaned 

• Biological safety cabinet (if applicable) 

• Eyewash 

• Inward airflow (desirable)

Biosafety Practices and 

Procedures

Hierarchy of Controls 

• Administrative Control 

• Engineering Control 

• Work Practices 

• Personal Protective Clothing or Equipment

Administrative Controls 

• Substitution 

• Authorization/Approval 

• Written biosafety procedures required for the 

experimental procedures and equipment including 

inventory of biological agents or materials 

• Laboratory personnel biosafety training 

• Medical Surveillance (BSL 2 and above) 

Health history 

Medical screening 

Immunization 

Serum storage 

Post-exposure prophylaxis

Engineering Controls 

• Biological safety cabinets, glove boxes 

• Animal containment caging systems 

• Safety equipment (filtered or sealed 

equipment) 

• Ventilation system 

• Containment facilities

Personal Protective Clothing and 

Equipment 

• Provides barrier against skin, mucous 

membrane or respiratory exposure to 

infectious agents during procedures 

• Prevent spread of contamination 

• Does not eliminate the hazard 

• Integrity wanes with use (i.e., change gloves 

frequently)

BSL 1: Work Practices and 

Procedures 


Non-infectious agent and tissue culture, media 

preparation 

• Prevent Cross Contamination 

Keep cultures covered 

Flame instruments and containers 

Use sterile media and equipment 

Keep hands or face away from cultures


Procedures 

• Biosafety Procedures 

Work with agents may be conducted on open 

bench 

Wash hands often 

No mouth pipetting 

No eating or drinking in lab 

Minimize aerosol generation 

Decontaminate work surfaces 

Wear applicable PPE


Procedures 

• Increasing emphasis on safety 

procedures and practices 

• Increasing need for staff training 

• Increasing need for competent 

supervision 

• Biohazard sign posted at entry door 

• Biohazard labels affixed on regulated 

waste containers 

• Use of personal protective equipment 

as a barrier to exposure: lab coat, 

gloves, eye and face protection 

• Some work on open bench allowed


Procedures 

• Aerosol generating procedures 

performed in a biosafety cabinet: 

Homogenizing 

Vortexing 

Vigorous mixing 

Pipetting infectious liquids 

Sonication 

Pouring 

• If breach occurs: 

Evacuate lab, post spill sign 

With appropriate PPE and 

disinfectant, decontaminate 

centrifuge, buckets, other 

items or areas

Correct Use of Biosafety Cabinets 

• Start Up 

 

 

 

 

Turn off ultraviolet light (if so 

equipped) as soon as you enter the 

room. 

Turn on all blowers and BSC 

illumination lights. 

Allow five minutes of operation to 

purge system; check flow alarm 

system audio and visual alarm 

function (if so equipped). 

Decontaminate readily accessible 

interior surfaces and items with a 

disinfectant (appropriate for the 

agents or suspected agents present) 

before loading and wait at least 10 

minutes prior to start of work.


• During Use 

Load supplies prior to 

work. 

Do not overload cabinet. 

Separate clean and dirty 

side. 

Work in center of work 

area. 

Do not block front or rear 

grills. 

Minimize disruption of 

airflow (turbulence). 

Clean up spill promptly. 

Discard waste within the 

cabinet.


• Shut Down 

Decontaminate and remove all 

items from interior work area. 

Decontaminate readily 

accessible interior surfaces with 

a disinfectant appropriate for 

the agents or suspected agents 

present. 

Turn on ultraviolet light (if so 

equipped). 

Allow five minutes of 

operation to purge system. 

Then wait at least 10 

minutes. 

Turn off BSC blower.


• Moving/Installation 

BSCs must be decontaminated prior to moving. In order to 

ensure filter integrity, the equipment must be recertified after 

the BSC is installed at its final new location. Arrangements 

need to be made well in advance in order for contractors to 

meet your schedule. The PI is responsible for contacting the 

contractor or to schedule this work. 

• Decontamination 

Decontamination is usually performed by certified 

professionals. 

• Certification 

All BSCs that are used for handling biohazardous materials 

must be recertified annually. SDSU has contracted with a 

specific contractor to provide a consistent level of 

certification and maintenance service. Contact EHS at (619) 

594-6778 to obtain contractor information.


– Open Flames 

• DO NOT use Bunsen burners or open flames 

Fire hazard 

Can damage HEPA filter 

Interferes with proper air flow 

Microincinerator preferred 

Burner with pilot light not a good alternative 

Open flames react with disinfectants (flammables)

Safe Use of Centrifuge 

• Use sealed tubes, rotors, and safety cups/buckets that are sealed d with 

O-rings 

• Inspect tubes, O-rings O 

and rotors for wear, and buckets for cracks, 

chips, erosion, etc. 

• Do not use aluminum foil to cap centrifuge tubes 

• Clean and maintain gaskets and O-ringsO 

• Change O-rings O 

if compromised 

• Load/unload centrifuge tubes, rotors and accessories in BSC 

• Small, low speed centrifuges may be placed in a BSC; however, high 

speed centrifuges pose additional hazards 

• Do not overfill tubes 

• Balance buckets, tubes and rotors properly before centrifugation 

• Wait 5 minutes (or 30 mins. for high speed centrifuge) after each h run 

before opening 

• Do not decant or pour off supernatant. Use a vacuum system with 

appropriate in-line reservoirs and filters

Safe Use of Blenders 

• Avoid use of glass blender jars, unless covered 

with polypropylene jar 

• Place disinfectant-moistened towel over the top of 

the blender during use 

• Before opening the blender jar, allow the unit to 

rest at least 1 minute for aerosols to settle and then 

open in a BSC 

• Decontaminate promptly after use

Minimizing Aerosols 

• Use careful pipetting practices 

• Avoid drops onto hard surfaces 

• Wipe up spills promptly with appropriate 

disinfectant 

• For ejection of liquid from micropipette 

No blowout 

No pressure ejection 

Use wall contact 

• Use capped tubes when mixing, blending, or 

vortexing 

• Pour liquids carefully 

• Avoid bubbles

Careful Pipetting Techniques 

• Never blow out last drop in 

pipette 

• Use pipetting aids with filters 

• Never mix by suction and 

expulsion (mix by sonication) 

• Discharge liquid down side 

of container, using tip-to 

to-wall 

contact 

• Deliver as close as possible to 

contents 

• Work over plastic-backed 

absorbent matting (ensure it 

doesn’t t slide forward or 

backward blocking air grill)

Use Extreme Care with Sharps 

• Use sharps if only absolutely 

required as part of a process 

• Percutaneous exposure risk 

Employ safe work practices 

Utilize safe sharp devices 

• Aerosol exposure risk 

Use biosafety cabinet for 

removal of air from needle 

• Use mechanical methods for 

needle removal 

• Never bend, recap or manipulate 

sharps by hand 

• Keep hands away from needle

Vacuum System Protection 

• In-line filter and disinfectant in collection and 

overflow flasks

Signs and Labels

Biohazard Signs 

• Completely filled out biohazard sign must be 

displayed at entrances to laboratories where 

biohazards are present and tissue culture rooms 

• Features fluorescent orange-red red sign with lettering 

“biohazard” and the international biohazard symbol 

in contrasting color, name of biohazardous agent or 

material, special entry requirement and PI and BSO 

contact number 

• Available free of charge from EHS, 

call (619) 594-2865

Biohazard Label 

• Must be attached to containers of biohazards or 

biohazardous waste containers. 

• Features fluorescent orange-red red label with 

lettering “biohazard” and the international 

biohazard symbol in contrasting color. 

• Red bags and sharps containers must also have the 

generator address label affixed prior to use: 

San Diego State University 

5500 Campanile Drive 

San Diego, CA 92182 

(619) 594-6778

Laboratory Moves

It is important that the laboratory is safe: 

• For custodians to clean 

• For contractors to work in 

• For the next group of laboratory personnel 

to occupy

Disposal and Decontamination of 


In preparation for moving, observe the following 

guidelines: 

• Label ALL biohazardous waste red bags and 

sharps container 

• Dispose of all biohazardous waste red bags and 

sharps containers at approved accumulation sites 

• EHS will not pick-up biohazardous waste except 

in unusual situations 

• Chemically disinfect biohazardous waste 

• Autoclave liquid biohazardous waste and dispose 

of down the drain

Moving the Biosafety Cabinet 

• Disinfect all BSC work surfaces prior to 

moving the BSC to a new facility. BSCs 

used for work with pathogenic organisms 

may require paraformaldehyde 

decontamination before being moved. 

• Each BSC must be recertified for correct 

airflow and filter integrity after it has been 

moved and placed in its final location.

Moving or Disposing of 

Refrigeration Units 

• Clear all materials stored inside the 

refrigeration units. 

• Disinfect all refrigeration units prior to 

removal or disposal. 

• Obtain clearance notification from EHS 

prior to removal or disposal of refrigeration 

units.

Shipment and Transportation

To Whom are the Shipping and 

Transportation Regulations Applicable 

Under IATA, DGR apply to anyone who handles, 

offers for transport, transports dangerous goods or 

causes dangerous goods to be transported. 

Under DOT, HMR apply to each person who 

performs, or causes to be performed, functions 

related to the transportation of hazardous materials 

such as determination of, and compliance with, 

basic conditions for offering; filling packages; 

marking and labeling packages; preparing 

shipping papers; handling, loading, securing and 

segregating packages within a transport vehicle, 

freight or cargo hold; and transporting hazardous 

materials.

Emergency Response to 

Biological Incidents 

Response to Biological Spills in 

the Laboratory 

(Intentional or Accidental)

Exposure Management 

For splash to eyes, mucous membranes, or 

broken area of the skin 

• Irrigate eyes with clean water, saline or 

sterile irrigants 

• Flush splashes to mouth, nose, and broken 

area of skin with water

Exposure Management 

For needlesticks or cuts with human blood, fluids, 

infectious agents or antibiotic resistant organism 

• Flush needlesticks and cuts with soap and water 

• Get medical evaluation ASAP 

• Inform PI, BSO and health professional (required 

mandatory reporting of incident) 

• Public Health Service has recommendations for 

post-exposure follow-up

Spill Clean-Up 

You can clean-up a biological spill if: 

• You are aware of the hazards and clean-up 

procedures (training required) 

• There is no potential for personal or 

environmental damage 

• The appropriate spill clean-up equipment is 

available 

• One or two people can clean-up the spill 

thoroughly in less than an hour 

Note: Spill incident still needs to be reported to 

BSO/EHS. If spill is in gallons or liters, call 

BSO/EHS.

Biological Spill Clean-Up Kit- 

Basic 

• Nitrile gloves (double gloving), splash 

goggles, shoe covers 

• Small disposable broom with dustpan, tongs 

or forceps (for picking up sharps) 

• Paper towels or other absorbent in the lab 

• Sharps container and/or biohazard waste 

bags 

• Disinfectant agent suitable for the agents in 

the lab

Spill Clean-Up for BSL 1-2 

If there is a spill inside the biosafety cabinet: 

• Keep the BSC running during spill and clean-up to contain aerosol. 

• Place absorbent paper on spill and soak with disinfectant. 

• Allow 20 minutes of contact time. Wipe up spill, working from the e edges to 

the center. Clean spill areas with fresh paper towels soaked in disinfectant. 

• Disinfect the BSC interior and any other equipment in the BSC with 

disinfectant. 

• Discard contaminated disposable materials using appropriate biohazardous 

waste disposal procedures. 

• Place contaminated reusable items in biohazard bags or autoclavable pans 

before autoclaving. 

• Run BSC 10 minutes after clean-up before resuming work or turning BSC 

off. 

Note: If you are working in a BSC and the power went off in the room or the 

BSC fan stops blowing, IMMEDIATELY LEAVE THE ROOM.


If the spill is in the laboratory but outside the biosafety cabine 

• Call the BSO if the material is RG 2 or greater. 

• Clear area of all personnel. Wait at least 30 minutes for aerosol ol to settle 

before entering spill area. 

• Remove any contaminated clothing and place in biohazard bag to be b 

autoclaved. 

• Put on disposable gown, safety glasses and gloves. 

• Initiate cleanup with disinfectant as follows: 

Place dry paper towels on spill then layer a second set of disinfectant 

soaked paper towels over the spill. 

Encircle the spill with additional disinfectant being careful to minimize 

aerosolization while assuring adequate contact. 

Allow at least a minimum of 20 minutes contact time to ensure germicid 

rmicid 

action of disinfectant. Wipe up spill, working from the edges to the 

center. Clean spill areas with fresh paper towels soaked in disinfectant. 

infectant. 

Decontaminate all items within the spill area. 

Discard contaminated disposable materials using appropriate 

biohazardous waste disposal procedures.


If the spill is outside the laboratory, in transit: 

• To prevent a spill, transport labeled biohazardous 

material in an unbreakable, well-sealed primary 

container placed inside of a second unbreakable, 

lidded container (cooler, plastic pan or pail). 

• Should a spill occur in a public area, do not attempt 

to clean it up without appropriate PPE. 

• Secure the area, keeping all people clear of the 

spill. 

• Call the BSO to assist in the clean-up. 

• Stand by during spill response and cleanup activity 

to provide information and assistance.

Biosecurity

Biosecurity vs. Biosafety 

• Biosecurity refers to ensuring the security 

of biological materials to prevent theft, 

illicit use or release. 

• Biosafety focuses on reducing exposure to 

and release of biological materials. 

• Integrating biosecurity and biosafety 

programs is important for work with select 

agents.

Inspection

Inspection Elements 

• Laboratory Identification 

• Containment Facility/Equipment 

• Work Practices 

• Hazard Communication 

• Biohazardous Waste Handling 

• Laboratory Personnel

Principles and Practices of Biosafety - San Diego State University

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