DIYbio Safety Manual
Tito Jankowski
Meredith and I drafted a DIYbio safety manual a few weeks ago. It's a
set of 11 principles for every DIYbiologist. I would like to see this
become a short but broad list of guidelines combined with a link to a
real safety manual such as BSL-1 by NIH.
http://openwetware.org/wiki/DIYbio:Notebook/Safety_Manual_1.0
For example:
1. Having a lab accident is a great way to ruin a week's worth of work.
Edit or comment - What do you think of this idea?
Kay, what's involved in keeping with BSL-1? Is there a set of hard
rules that we could link to?
Tito
On Thu, Jan 8, 2009 at 1:35 PM, Splicer <black...@gmail.com> wrote:
>
> On Jan 8, 12:56 am, "Nick Taylor" <nick1...@googlemail.com> wrote:
>
>> Anyway... although safety is vitally important on a practical level, my
>> advice on a PR level is not to voluntarily discuss safety at all... rather,
>> dwell on the community/peer-review aspects - because by constantly deploying
>> the Safety Frame you're also invoking the Risk Metaphor.
>
>
> I agree. I think part of the reason why the safety/security topic
> keeps coming up is that it's something everyone can relate to and have
> an opinion about. Even as a newcomer when the rest of the art feels
> difficult. I know it's true for me.
>
> As for PR... I agree. If someone who doesn't know anything about SB
> asks about safety/security give them the 'big corporations are bad'
> speech. Talk about how patents hinder development, about patent
> gridlocks and so on. Give them that bag to chew on. Talk about how
> unknown grad students have built an arsenic detector which is
> accurate, costs pennies and can save millions of people from getting
> cancer. Meredith has the melamine angle on her project. Add to that
> 'big corp is greedy and bad', garage biology is underdog and good.
> Hint that this talk about safety/security is something big corp uses
> to stop garage biology in it's tracks so they can go on and do bad
> things.
>
> -Splicer
>
> >
>
Lora
http://www.ilpi.com/msds/index.html
On Jan 8, 4:50 pm, "Tito Jankowski" <titojankow...@gmail.com> wrote:
> Hi everyone,
>
> Meredith and I drafted a DIYbio safety manual a few weeks ago. It's a
> set of 11 principles for every DIYbiologist. I would like to see this
> become a short but broad list of guidelines combined with a link to a
> real safety manual such as BSL-1 by NIH.
>
> http://openwetware.org/wiki/DIYbio:Notebook/Safety_Manual_1.0
>
> For example:
> 1. Having a lab accident is a great way to ruin a week's worth of work.
>
> Edit or comment - What do you think of this idea?
>
> Kay, what's involved in keeping with BSL-1? Is there a set of hard
> rules that we could link to?
>
> Tito
>
Kay Aull
guidelines below. That's the NIH standard for handling non-pathogenic
organisms, such as E. coli K12 and lactobacillus. They're pretty
good, but don't cover the nuts and bolts of actual setup. For
instance, saying that "policies for the safe handling of sharps should
be instituted" is less useful than "here's a diabetic supply company
that will mail you a sharps container, and you mail it back when
full". We can capture that kind of information for the community's
benefit.
Link to biosafety guidelines: http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4s3.htm
Mackenzie Cowell
Mac
Recombinant DNA Home Lab Biosafety Manual
This manual describes appropriate facilities and procedures for an unaffiliated researcher, without access to institutional support of a Biosafety Office, Institutional Review Board, etc. The safe, legal, and socially acceptable limits for research done in this context are necessarily limited.
Household
Food grade
Work done at food grade begins with only materials which are sold as human food or beverages and which are safe and legal to consume. Procedures are those typical of normal cooking, and can be done in an ordinary kitchen. A variety of interesting chemistry and microbiology experiments are possible, often with edible results. The researcher can make bread, yogurt, sourdough, kefir, kombucha, beer, wine, distilled beverages, etc. DNA can be extracted (grind fresh food, soak in buffer (table salt, baking soda), filter, add 95% cold vodka and collect precipitate). Work with recombinant DNA is not possible. Risks are those typical of cooking, including cuts, burns, and illness resulting from consumption of inedible or spoiled food.
Household chemicals
A variety of useful household chemicals are unsafe for food but nevertheless may be commonly found in the house or kitchen. For example, sodium hydroxide (lye) is used in soapmaking, a variety of solvents are used in cleaning and painting, bleach, methylene blue is used in fishtanks. Despite the ubiquity of such chemicals, they should not be treated casually. One amateur soapmaker left a pitcher of concentrated lye on the kitchen counter after a late night of work. Her husband awoke early, and mistook it for water, with the predictable horrific consequence.
Biosafety Level 1
Level 1 is the basic level for doing recombinant DNA work.
NIH: "Biosafety Level 1 is suitable for work involving agents of unknown or minimal potential hazard to laboratory personnel and the environment. The laboratory is not separated from the general traffic patterns in the building. Work is generally conducted on open bench tops. Special containment equipment is not required or generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science ."
CDC biosafety manual 4th ed http://www.cdc.gov/OD/ohs/biosfty/bmbl4/bmbl4toc.htm
"Biosafety Level 1 practices, safety equipment, and facility design and construction are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. [...] Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for handwashing. "
Lab Design
Laboratory location and access
Separated from public areas by door.
Size of door openings to allow passage of all anticipated equipment.
Surface (i.e., floors, walls, ceilings, sealants) Finishes and Casework (all recommendations only, not requirements, according to PHA OLS)
Surfaces to be scratch, stain, moisture, chemical and heat resistant in accordance with laboratory function.
Surfaces to provide impact resistance in accordance with laboratory function.
Interior coatings to be gas and chemical resistant in accordance with laboratory function (e.g., will withstand chemical disinfection, fumigation).
Bench tops to have no open seams.
Bench tops to contain spills of materials (e.g., with marine edges and drip stops).
Benches, doors, drawers, door handles, etc. to have rounded rims and corners.
Backsplashes, if installed tight to wall, to be sealed at wall-bench junction.
Reagent shelving to be equipped with lip edges.
Drawers to be equipped with catches, i.e., to prevent the drawer from being pulled out of the cabinet.
Cabinet doors not to be self-closing.
No specific HVAC requirements or recommendations, but should nevertheless be adequate for personnel comfort. Note high humidity (>50%) helps pipetting precision, and ventilation can be useful in eliminating unpleasant smells.
No lighting mentioned, but again should be adequate for tasks at hand.
Containment perimeter
(Recommended) Autoclave or other acceptable means of waste treatment/disposal to be provided.
Windows, if they can be opened, to be protected by fly screens.
Laboratory Services (i.e., water, drains, gas, electricity, and safety equipment)
Hooks to be provided for laboratory coats at laboratory exit; street and laboratory clothing areas to be separated.
Handwashing sinks to be located near the point of exit from the laboratory or in anteroom.
Formal certification and commissioning is not required for a Level 1 lab.
NIH:
Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.
Laboratory furniture is sturdy.
Spaces between benches, cabinets, and equipment are accessible for cleaning.
Sources: http://www.phac-aspc.gc.ca/ols-bsl/lbg-ldmbl/index-eng.php
Biological Safety, Fleming and Hunt, ASM Press 2000 reviews global regulation, incl Canada.
WHMIS requires: 1) labels 2) MSDS 3) training
Canadian Environmental Protection Act (RSC 1985 c16 4th Suppl) req all new substances incl living organisms be assessed for potential to harm the environment.
New Substances Notification Regulation (SOR/94-260)
Federal Transportation of Dangerous Goods Act and Regulations
Edmonton sewer and sewer use bylaws: "any substance that is designated as a hazardous substance within the regulations of the Environmental Protection and Enhancement Act and the Waste Control Regulation of the Province of Alberta, and any successor Act or regulations; ", "biological waste" means waste from a hospital, medical clinic, health care facility, mortuary or biological research laboratory which contains or may contain:
(i) pathogenic agents that cannot be effectively mitigated by wastewater treatment; and
(ii) experimental biological matter that may be hazardous to human health or detrimental to the environment;"
Waste bylaw says no hazardous waste; no radioactive waste.
Lab Rules
Again quoting http://www.phac-aspc.gc.ca/ols-bsl/lbg-ldmbl/index-eng.php, section 3.3.1.
The following general practices are required for all laboratories handling infectious substances.
A documented procedural (safety) manual must be available for all staff, and its requirements followed; it must be reviewed and updated regularly.
Personnel must receive training on the potential hazards associated with the work involved and the necessary precautions to prevent exposure to infectious agents and release of contained material; personnel must show evidence that they understood the training provided; training must be documented and signed by both the employee and supervisor; retraining programs should also be implemented.
Eating, drinking, smoking, storing of either food, personal belongings, or utensils, applying cosmetics, and inserting or removing contact lenses are not permitted in any laboratory; the wearing of contact lenses is permitted only when other forms of corrective eyewear are not suitable; wearing jewelry is not recommended in the laboratory.
Oral pipetting of any substance is prohibited in any laboratory.
Long hair is to be tied back or restrained so that it cannot come into contact with hands, specimens, containers or equipment.
Access to laboratory and support areas is limited to authorized personnel.
Doors to laboratories must not be left open (this does not apply to an open area within a laboratory).
Open wounds, cuts, scratches and grazes should be covered with waterproof dressings.
Laboratories are to be kept clean and tidy. Storage of materials that are not pertinent to the work and cannot be easily decontaminated (e.g., journals, books, correspondence) should be minimized; paperwork and report writing should be kept separate from such biohazardous materials work areas.
Protective laboratory clothing, properly fastened, must be worn by all personnel, including visitors, trainees and others entering or working in the laboratory; suitable footwear with closed toes and heels must be worn in all laboratory areas.
Where there is a known or potential risk of exposure to splashes or flying objects, whether during routine operations or under unusual circumstances (e.g., accidents), eye and face protection must be used. Careful consideration should be given to the identification of procedures requiring eye and face protection, and selection should be appropriate to the hazard.
Gloves (e.g., latex, vinyl, co-polymer) must be worn for all procedures that might involve direct skin contact with biohazardous material or infected animals; gloves are to be removed when leaving the laboratory and decontaminated with other laboratory wastes before disposal; metal mesh gloves can be worn underneath the glove.
Protective laboratory clothing must not be worn in nonlaboratory areas; laboratory clothing must not be stored in contact with street clothing.
If a known or suspected exposure occurs, contaminated clothing must be decontaminated before laundering (unless laundering facilities are within the containment laboratory and have been proven to be effective in decontamination).
The use of needles, syringes and other sharp objects should be strictly limited; needles and syringes should be used only for parenteral injection and aspiration of fluids from laboratory animals and diaphragm bottles; caution should be used when handling needles and syringes to avoid auto-inoculation and the generation of aerosols during use and disposal; where appropriate, procedures should be performed in a BSC; needles should not be bent, sheared, recapped or removed from the syringe; they should be promptly placed in a puncture-resistant sharps container (in accordance with Canadian Standards Association [CSA] standard Z316.6-95(R2000))(6) before disposal.
Hands must be washed after gloves have been removed, before leaving the laboratory and at any time after handling materials known or suspected to be contaminated.
Work surfaces must be cleaned and decontaminated with a suitable disinfectant at the end of the day and after any spill of potentially biohazardous material; work surfaces that have become permeable (i.e., cracked, chipped, loose) to biohazardous material must be replaced or repaired.
Contaminated materials and equipment leaving the laboratory for servicing or disposal must be appropriately decontaminated and labelled or tagged-out as such.
Efficacy monitoring of autoclaves used for decontamination with biological indicators must be done regularly (i.e., consider weekly, depending on the frequency of use of the autoclave), and the records of these results and cycle logs (i.e., time, temperature and pressure) must also be kept on file.
All contaminated materials, solid or liquid, must be decontaminated before disposal or reuse; the material must be contained in such a way as to prevent the release of the contaminated contents during removal; centralized autoclaving facilities are to follow the applicable containment level 2 requirements.
Disinfectants effective against the agents in use must be available at all times within the areas where the biohazardous material is handled or stored.
Leak-proof containers are to be used for the transport of infectious materials within facilities (e.g., between laboratories in the same facility).
Spills, accidents or exposures to infectious materials and losses of containment must be reported immediately to the laboratory supervisor; written records of such incidents must be maintained, and the results of incident investigations should be used for continuing education.
An effective rodent and insect control program must be maintained.
NIH generally similar; minor leniencies include: food storage allowed (if separate fridge), facilities/protective clothing "shall be provided that are appropriate for the risk of exposure"; adds "All procedures are performed carefully to minimize the creation of aerosols." CDC: "All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility. "
A common decontamination practice is to mix the culture with bleach and let sit before pouring down the drain. Full-strength household bleach is not a hazardous waste and is an effective disinfectant.
Type of work permitted
The NIH recombinant dna guidelines (http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html) covers all US recombinant dna research done at facilities accepting NIH funding, and research abroad supported by the NIH. "Recombinant DNA molecules are defined as either: (i) molecules that are constructed outside living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above". Even if the NIH guidelines do not legally apply to you, you may nevertheless wish to comply with them as if they did. Unfortunately, compliance in general is not straightforward for nonaffiliated researchers, since it involves various notifications/approval by the institution's review board, biosafety office, in concert with the NIH. This third-party review forms a vital role in general biosafety, since it acts a check, with the reviewers being expected to be more dispassionate about the true risks of a research plan than the plan's proposer. Additionally, and not irrelevantly from an institution's point of view, such a structure spreads responsibility in the event something goes wrong.
While this system works well and is appropriate in general, it is possible to identify classes of experiment for which the risks are minimal, and for which the full institutional review process is bureacratic overkill. The NIH guidelines
Much better to be exempt.
Section III-F. Exempt Experiments
The following recombinant DNA molecules are exempt from the NIH Guidelines and registration with the Institutional Biosafety Committee is not required:
Section III-F-1. Those that are not in organisms or viruses.
Section III-F-2. Those that consist entirely of DNA segments from a single nonchromosomal or viral DNA source, though one or more of the segments may be a synthetic equivalent.
Section III-F-3. Those that consist entirely of DNA 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.
Section III-F-4. Those that consist entirely of DNA 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).
Section III-F-5. 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. A list of such exchangers will be prepared and periodically revised by the NIH Director with advice of the RAC after appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), Major Actions). See Appendices A-I through A-VI, Exemptions Under Section III-F-5--Sublists of Natural Exchangers, for a list of natural exchangers that are exempt from the NIH Guidelines.
Section III-F-6. Those that do not present a significant risk to health or the environment (see Section IV-C-1-b-(1)-(c), Major Actions), as determined by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public comment. See Appendix C, Exemptions under Section III-F-6 for other classes of experiments which are exempt from the NIH Guidelines.
Appendix A-I. Sublist A
Genus Escherichia
Genus Shigella
Genus Salmonella - including Arizona
Genus Enterobacter
Genus Citrobacter - including Levinea
Genus Klebsiella - including oxytoca
Genus Erwinia
Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas mendocina
Serratia marcescens
Yersinia enterocolitica
Appendix A-II. Sublist B
Bacillus subtilis
Bacillus licheniformis
Bacillus pumilus
Bacillus globigii
Bacillus niger
Bacillus nato
Bacillus amyloliquefaciens
Bacillus aterrimus
Appendix A-III. Sublist C
Streptomyces aureofaciens
Streptomyces rimosus
Streptomyces coelicolor
Appendix A-IV. Sublist D
Streptomyces griseus
Streptomyces cyaneus
Streptomyces venezuelae
Appendix A-V. Sublist E
One way transfer of Streptococcus mutans or Streptococcus lactis DNA into Streptococcus sanguis
Appendix A-VI. Sublist F
Streptococcus sanguis
Streptococcus pneumoniae
Streptococcus faecalis
Streptococcus pyogenes
Streptococcus mutans
What's Level 1 organism?Appendix C-II. Escherichia coli K-12 Host-Vector Systems
Experiments which use Escherichia coli K-12 host-vector systems, with the exception of those experiments listed in Appendix C-II-A, are exempt from the NIH Guidelines provided that: (i) the Escherichia coli host does not contain conjugation proficient plasmids or generalized transducing phages; or (ii) lambda or lambdoid or Ff bacteriophages or non-conjugative plasmids (see Appendix C-VII. Footnotes and References of Appendix C, Footnotes and References of Appendix C) shall be used as vectors. However, experiments involving the insertion into Escherichia coli K-12 of DNA from prokaryotes that exchange genetic information (see Appendix C-VII. Footnotes and References of Appendix C, Footnotes and References of Appendix C) with Escherichia coli may be performed with any Escherichia coli K-12 vector (e.g., conjugative plasmid). When a non-conjugative vector is used, the Escherichia coli K-12 host may contain conjugation-proficient plasmids either autonomous or integrated, or generalized transducing phages. For these exempt laboratory experiments, Biosafety Level (BL) 1 physical containment conditions are recommended. For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the host organism unmodified by recombinant DNA techniques; the Institutional Biosafety Committee can specify higher containment if deemed necessary.
Appendix C-II-A. Exceptions
The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) experiments involving the cloning of toxin molecule genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).
Appendix C-III. Saccharomyces Host-Vector Systems
Experiments involving Saccharomyces cerevisiae and Saccharomyces uvarum host-vector systems, with the exception of experiments listed in Appendix C-III-A, are exempt from the NIH Guidelines. For these exempt experiments, BL1 physical containment is recommended. For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the host organism unmodified by recombinant DNA techniques; the Institutional Biosafety Committee can specify higher containment if deemed necessary.
Appendix C-III-A. Exceptions
The following categories are not exempt from the NIH Guidelines: (i) experiments described in Section III-A which require Institutional Biosafety Committee approval, RAC review, and NIH Director approval before initiation, (ii) experiments described in Section III-B which require NIH/OBA and Institutional Biosafety Committee approval before initiation, (iii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iv) large-scale experiments (e.g., more than 10 liters of culture), and (v) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).
"Risk Group 1 (RG1) Agents that are not associated with disease in healthy adult humans. A strain of Escherichia coli (see Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen); and (2) does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors."
Local regulations
lab glassware is controlled in texas (wikipedia article on erlenmeyer flask).
plants incl cyanobacteria are tightly regulated in Hawaii
Cambridge MA regulates rDNA, based on NIH http://www.cambridgepublichealth.org/services/regulatory-activities/rdna/overview.php
Positive and negative anecdotes
Steven Kurtz http://www.wired.com/medtech/health/news/2004/06/63637
Victor Deeb: http://www.telegram.com/article/20080809/NEWS/808090323/1008/, http://pubs.acs.org/cen/science/86/8645sci1.html
Molecular Probes started in a kitchen in 1975 http://en.wikipedia.org/wiki/Molecular_Probes
Other small companies -- eg AltaZymes, Ambi Products, thousands of others
Peter Mitchell received the 1978 Nobel Prize in Chemistry for discovering chemiosmosis, work done as an independent researcher http://en.wikipedia.org/wiki/Peter_D._Mitchell
Strategies to help ensure societal acceptance of your work
Code of ethics
Clear goals
Professional-looking work space
Professional work habits, eg lab notebook practice
Acquire formal certifications
Get a local business license
Work with others
Do it as a day job
Sharing your work
Legalities, open source licenses
http://opensource.org/docs/osd
Note OSD prohibits discrimination by field of endeavor, so for example "For research only", or "For non-commercial use only" restrictions would violate the OSD. Unfortunately, different fields of endeavor are treated very differently by regulators, so there can a conflict between "open source" principles and easy sharing of your work.
Practicalities -- safely, legally and cheaply shipping dna
FAQs
Can I eat, drink, or otherwise consume anything produced in my laboratory?
Okay, but how about if I feed it to my cat? Or my cricket?
Okay, but can I plant it in my backyard?
How can I share my work with others?
How should I proceed if my work can't be done under these restrictions?
And if I don't, what laws would I be breaking?
Bibliography
SR Rayburn, The Foundations of Laboratory Safety: A Guide for the Biomedical Laboratory, 1990
Miller et al LABORATORY SAFETY: PRINCIPLES AND PRACTICES, ASM.
Jose-Luis Sagripanti and Aylin Bonifacino, "Bacterial Spores Survive Treatment with Commercial Sterilants and Disinfectants," Appl. Environ. Microbiol. 65, no. 9 (September 1, 1999): 4255-4260.
http://www.ilpi.com/msds/index.html Free online MSDS