Team:Paris Bettencourt/Safety

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Contents 1 Safety 2 Our Work Environment 2.1 Research Unit U1001 2.2 Supervision 3 Risk assessment for our project 3.1 Organisms used 3.2 References for risk assessment 4 Risk management in our daily work

Safety

We are following the official iGEM safety instructions as well as the updated questions sent to us.

Our Work Environment

Research Unit U1001

We are working in the [http://www.necker.fr/tamara/pages/people.html U1001 research unit] which is shared between INSERM (National Institute of Health and Medical Research) and [http://app.parisdescartes.fr/cgi-bin/WebObjects/Labs.woa/wa/showInfoLabo?cle=20101353#ficheLabo Paris Descartes University]. It is fully equipped for bacterial genetics and molecular biology work in terms of material and people having the competence to do the research. The lab is monitored on regular basis by security officers of both the Paris Descarteds University and the INSERM.

Supervision

Our team is under daily supervision of primarily Ariel Lindner Senior Research Scientist in U1001. We receive regular help as well from Chantal Lotton (Engineer and Hygiene and Security Correspondant) as well from two Ph.D students Yifan Yang and Antoine Decrulle, both working on site. The whole lab team is also here at times to explain and advise us.

Risk assessment for our project

Organisms used

Bacillus subtilis is a gram-positive bacteria, rod-shaped, aerobic, sporulating ( very resistant spores: heat, dessication etc.). It is ubiquitous in the environment and it is not usually a human pathogen. It is considered a Level 1 Biosafety Containment Agent which goes according to the following definition: "Agents of no or minimal hazard under ordinary conditions of handling" [http://www.unm.edu/~sheaweb/sheamanual/biosfty/BIOSAFA-pg3.01-A7only.htm]. However, we are conscious that as any bacterium it can be dangerous and here is a short listing of symptoms and how to cure them: All traumatic wounds, infected burns and any serious lesions can potentially be a terrain for Bacillus strains but it is VERY rare. When found, subtilis is usually mixed with other types of bacteria, it can cause a bacteremia but it is easily eradicated.
furthermore, hypersensitivity reactions have been observed in the litterature with contact to autolysates. Symptoms: asthma, skin inflammation (dermatitis), lung inflammation (pneumotitis).

• TREATMENT: All Bacillus species can be treated with non-ß-lactam antibiotics
  

Escherichia coli: we use DH5a and K12?
E. coli is also considered as a Level 1 Biosafety Containment agent. As already mentionned, our lab is especially equipped for microbial manipulation and everything is done to avoid risk to the personnel or release to the environment.

References for risk assessment

1. Farrar WE Jr. 1963. Serious Infections Due to “Non-Pathogenic” Organisms of the Genus Bacillus. Am J Med. Vol:34. pp:134-41 [http://www.sciencedirect.com/science?_ob=MiamiImageURL&_imagekey=B6TDC-4BXGKV2-1B4-1&_cdi=5195&_user=658968&_pii=0002934363900470&_check=y&_origin=&_coverDate=01%2F31%2F1963&view=c&wchp=dGLzVlz-zSkzS&md5=5378af9f1b74b03445c94b3a24f99ac6&ie=/sdarticle.pdf PDF ]
2. Farrar WE , Reboli AC. 2006. The Genus Bacillus-Medical. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, editors. Prokaryotes, Part 1, Section 1.2. New-York: Springer. 609-630 [http://www.springerlink.com/content/mp23l6460306188q/fulltext.pdf PDF]
3. Turnbull, PCB. 1996. Bacillus. In: Baron S, editor. Medical Microbiology. 4th edition Chapter 15. Galveston (TX): University of Texas Medical Branch at Galveston. [http://www.ncbi.nlm.nih.gov/books/NBK7699/]
4. Bacillus on Wikipedia [http://en.wikipedia.org/wiki/Bacillus]
5. Bacillus subtilis Final Risk Assessment [http://epa.gov/biotech_rule/pubs/fra/fra009.htm]

Risk management in our daily work

France, as a member of the European Union follows the Directives of the European Parliament (such as this one [http://www.bmwf.gv.at/fileadmin/user_upload/forschung/gentechnik/2009-41-EC.pdf Directive 2009/41/EC on the contained use of genetically modified micro-organisms]), we and the research unit we work in apply the general guidelines that are outlined notably in the aforementioned Directive.

The following are basics in Molecular Biology labs that were presented to us during a big laboratory meeting prior to the start of the experiments.
We acknowledge that these rules or principles might be at times specific to our laboratory and to our staff/members but they are generally relevant.

The information about biosafety in the lab presented here have been gathered during the introduction made by the Correspondant of Hygiene and Security ([http://www.necker.fr/irnem/units/search.php?iduser=1950&pass=1 Chantal Lotton]) to the safe use of each instrument and machine as well as general guidelines of good lab practice. The handling of waste, how to keep things sterile, how to use the hood etc. was also explained to us
In our team, there are also two designated lab managers that are notably in charge of knowing the functioning of each apparatus and of the security issues; team's students had, as a mandatory part of their undergraduate curriculum, a Hygiene and Security course. In case any problems arise Chantal Lotton, the lab's security officer, can be contacted 24H/24H to seek help and/or advice.

There are basic precautions and security procedures that MUST be followed in any laboratory.

• The ten general rules :

Rule n°1: If you're not sure of anything: ASK !
Rule n°2 : Don't smoke or eat or drink in the lab.
Rule n°3 : Don't wear scarves and tie your hair back if needed. Wear coat when it’s not so hot.
Rule n°4 : Don't leave any personal belongs (phone...) because of thiefs.
Rule n°5 : Don't work ALONE at night and during the weekend (and inform the on-call guard of your presence).
Rule n°6 : Be careful, products may be harmful even if they seem inoffensive.
Rule n°7 : Identify your stuff (iGEM + date + if sterile) and CLEAN UP once you've finished.
Rule n°8 : Tell us if something is running out so that we can buy them in advance.
Rule n°9 : Close both lab doors before leaving.
Final rule : Turn off all the machines before leaving (especially the bain marie, 
because of fire and surtension problems in this lab!). Check that -20°C and -80°C are OK every morning.

If you've received chemical spatter on your eyes or your skin, use ONLY water to clean them (at least 10 min under the water)! No detergent!

• Trash can :

Solid carton: Press down and compress!
Black: For everyday use – paper, plastic, enveloppe...
Cardboard yellow box: Biological stuff (Petri dish closed, pipette on verticle angle)
To close the box: Read the instructions on the box, identify it (U1001 + date), then put it in the hallway
Plastic yellow box: Sharp/ cutting items (needles, glass...)

Liquid : Methanol and other: chemical waste bottle

• BET bench :

ASK Chantal before using - Cancer Risk (UV and BET)!
Wear nitrile blue or purple glove, long-sleeved blouse and goggles
No BET in electrophoresis, only in tray.
Use only dishes, pipettes and trash can for BET.
Buffer : 0,5X (be careful if 1X = crystal) TBE (or TAE)

Specials trashes for EtB: Solid (gel, gloves, contamined stuff) in big white box (under the bench)
Tips in yellow box on the bench “EtB”
Liquid: in 5L plastic bottle on the bench (decontamination with 2 bags of charcoal)

• Lavery :

In Dirty stuff Box (Buffer …): Rinse bottle and take out scotch
In Contaminated stuff Box (bacteria …): Let it in the box. (It will be wash with Javel water)
If you decontamined with Javel water, wear blouse.

• Microscopy :

ASK MING for a briefing before using! (his holidays: 2-17 of July)
Verify schedule before entry in the room. Do not let the door open (night for experiments which are on, air conditioned room)

• Machines instructions :
  

Autoclave :
To be used only by COMPETENT AND AUTHORIZED PEOPLE : Kevin and Daniel < risk of burning and glass breaking.

Always UNSCREW the bottles before introducing them into the machine!
Do not try to open during each cycle. Check temperature (85°C) and pressure of autoclave (1 bar) before opening.
Be careful, the top is very hot (high risk of burning yourself, 85°C when opened): Use mittens to take out the sterilized bottles! Check if the scotch lines turned black.
If it's LBA bottle, mix it after sterilizing.

Micro-waves :
Always UNSCREW the bottles before introducing them into the machine!
BIG RISK OF BURNING: Use mittens to take out the bottles!

Washing machine :
To be used only by competant and authorized people (Kevin and Daniel) < risk of burning and glass breaking.
Check the level of water before use (the reserve as to be full). Do not try to open during each cycle.

Osmosed water recipient :
Be careful to close it well (90°) to avoid flood.

Precision balance :
Prepare what you need and close doors everytime before weighing. Clean after use.

Shaker-Incubator :
Rack has to be stable (check that black screws are on tight)
Don't hesitate to launch a new one if necessary.
Sticky band : don't put your hand on it (there is polystyrene stuff on it). To wash it, use only water + soap.
Always incline your falcon and unscrew it a little (to let the air go inside)

37°C and 30°C Incubators :
Be careful when closing the door, it is fragile !
Petri dishes with gelose has to be put on the top (to avoid deshydration)

Hood :
It only protects the area under the hood and not us ! NO PATHOGEN INSIDE!
Clean your hands. Clean the work area with 70% ethanol BEFORE and AFTER use.
(ethanol in 5L bottle (kitchen) + osmosed water)
Try to occupate only middle of area in order to share with someone else.

Centrifuges :
Don't forget to put the hat and to BALANCE out your tubes (weight and symmetric position).
Once you've finished, shut the machine down. Leave the top open.

Freezers :
Use the -80°C only for long term stocks. Use -20°C for working stocks.
Don't leave the door open when you looking for a strain in the boxes.
Close the door of the “kitchen”: this room is air conditioned because of the -80°C!
Use cardboard boxes for storage (not plastic racks).
Always check the temperature and if there is a problem of cut, do NOT open the door (years of research are inside...)

If there is any problem: 2255 (technical service of the faculty: you will join Jean Marie / Florent electricians or Eric (boss)).

4°C - Powder of IPTG, Antibiotics … : Use glove

Burner :
Sterilizes an area of 30 cm radius around the burner. Don't speak when working within the sterile zone, otherwise you'll spread your bacteria !
Be careful at your hear

Bain Marie :
DO NOT FORGET to turn it off once you're done (high risk of combustion!)

PCR / Thermocycler :
Don't let it work at 4°C overnight as much as possible (electricity surtension)! Shut it down after use.

Biophotometer :
1/10 dilution in 50 microL. Do not forget to shut down and close the chamber with the black cap.

Spectrophotometer and Tecan :
Turn them off before leaving.