P1 SOP

SOP in the Lab - Standard Operating Procedure in English

Introduction:
This document describes the standard operating procedure (SOP) for activities in Hackuarium’s P1 lab. It covers behavior, safety, storage and protocols that are to be followed in the lab space. Each user has to read the procedure and approve it in order to participate in any kind of activities within the space. This document was begun by Yann Pierson (thanks!!) and inspired by the World Health Organization. Laboratory biosafety manual. – 3rd ed

Behavior in the P1 laboratory:
The lab is a small space, a maximum of six collaborators can perform manipulations at any time. Another collaborator can enter only to supervise, request or take an item of his own. Collaborators have to communicate and avoid any collisions. A calm and relaxed state of mind has to be observed when performing activities. Utmost cleanliness is essential after and during any work. Benches have to be cleaned after manipulations, with 70% ethanol and should be spotless to avoid any contamination. The material in the lab should generally stay there for lab use, and not be taken into the rest of the Hackuarium (biohacker space). It is strongly encouraged to thoroughly wash hands when experiments are over. Food and beverages are strictly forbidden.
See this page first, which lists basic rules, if you would like to do P1 experiments at Hackuarium, and talk to the Biosafety Officer (Rachel) or the deputy (Javier)!

Safety:
Personal protective equipment (PPE):
Each collaborator is expected to use the necessary safety equipment when working in the P1 laboratory. All are located at the entrance: safety goggles & lab coats of various sizes. In the lab gloves can be found in easily accessible locations. Safety goggles and lab coats are to be worn at all times, gloves are case dependant but are strongly recommended. Closed shoes are to be worn.
Spillage:
Dropping a vial containing liquid or a flask of powder is common. In a lab if not properly handled a spill can have dramatic effects. Here we detail the SOP for the different types of spills that happen with their respective anti-spill kits: acidic, basic, contaminated and neutral spills.
General liquid restrictions:
Technically the P1 lab space does not allow use of flasks over a volume of 500ml (incubator volume). A culture grows well at a fifth of this total volume (oxygenation requisite), which theoretically makes it 100ml culture. Safety wise, using a rather small enclosed space, spills have to stay manageable. To avoid bacterial spread outside of the P1 lab a maximal volume of populated medium of 200ml per flask is allowed. A maximal volume of 1L in total, populated or sterile medium, is allowed. A maximal volume of 1L per buffer is allowed. No strong acids or bases are allowed in the P1 laboratory.
Content of spill kit:

• Thick gloves

• Absorbent

• pH paper

• Base neutralisation

• Acid neutralisation

• Disinfectant

• Plastic Bags

• Scoop & scraper

Procedure:
1. Alert your collaborators.
2. If corrosive material landed on your lab coat or clothing, remove them.
3. If corrosive material landed on your skin or eyes, go and wash under abundant water for 15min. Leave the rest to your collaborators. Call emergency if situation is out of control.
4. Unplug and turn-off all nearby electrical equipment.
5. If no direct danger is apparent (all electronics unplugged, no source of fire), take time to consider most appropriate actions and the procedure to be used. Don’t overcrowd the spill zone.
6. Take the spill kit located above the waste zone and open it in a clear zone.
7. If glass was broken wear anti-cut gloves (green gloves).
8. Follow the procedure describing your situation:
Neutral liquid:
Your solution was not containing any growing bacteria, was not an acid below pH 5 or basic above pH 10. This case is typical for buffers, or growth medium, freshly prepared.
If glass was broken, collect the pieces and put them in the glass trash.
Scoop the liquid using the scraper. Empty in liquid trash container.
You can use absorbent paper or sand for the residual liquid and trash it in the solid waste container.

Neutral Solid:
You dropped a solid which is not a base (ex: NaOH) or an acid.
If glass was broken, collect the pieces and put them in the glass trash.
Scoop the solid using the dust pan. Empty in solid trash container.
Use reasonable amounts water to rinse and clean. Trash papers in solid trash container.

Acidic liquid: Put on thick safety gloves, face mask and lab coat.
Take the acid neutralizing solid and pour it over the spill from outside to inside. It should bubble.
Test with pH paper until it goes above pH 5.
Proceed to clean as in the neutral liquid section (keep gloves on!).

Basic Liquid:
Put on thick safety gloves, face mask and lab coat.
Take the base neutralizing solution and spray it over the spill from outside to inside.Test with pH paper until it goes below pH 10.
Proceed to clean as in the neutral liquid section (keep gloves on!)

All cases of spillage have to be announced on the Slack in the biolab section by mentioning the type of spill and the material that was used and is now empty.

Biowastes:
At the left of the entrance is located the waste zone. The wastes are organized over a big white tray that can contain the wastes if they happened to break.

Liquid wastes:
A 5L bottle is used to contain the liquid wastes. All the liquids used in the P1 enclosure have to be poured in this container. The container itself should be in a safe place (maybe in the fume hood to start!) or perhaps attached to the nearby bench in order for it not to fall. A funnel is placed next to it to ease pouring. Once the liquid reaches the 5L mark the bottle has to be filled with 400ml commercial bleach and left for 30 min in order to deactivate all microorganisms. The bottle can be emptied in the drain and rinsed.

Solid wastes:
The solid wastes (non perforant material such as needles, shards or items that might damage the bag) are to be disposed after disinfection in the normal waste for incineration. in the closed container containing the biowaste bag. Once the container is full, close the bag with a plastic clamp. In doubt of damaged bag, you can put it in a second biowaste bag. Autoclave the bag for 30 min at 121°C. The autoclaved bag has to be placed in a normal trash bag, closed with a plastic clamp and trashed via the usual elimination route. In Ecublens, no big autoclave is available for such a method. We inactivate GMOs, generally by bleach treatment, for safe disposal in the garbage, which is furthermore incinerated in Switzerland.

Potentially dangerous instruments:
Plugs:
All the multiplugs have to be fixed above ground level or above the working bench to minimize risks of electrocution.
Centrifuges:
Centrifuges if not correctly balanced when running might cause deadly damages. Tubes have to be balanced with a counterweight of a maximal weight deviation of 0.2 grams. Certain centrifuges have indications on their lids or on the rotors the user needs to follow. Rotors max speed are not to be exceeded.
Microwaves:
When heating up bottles in the microwave ensure the cap is open to avoid overpressure and explosion. Wear gloves when taking the item out as it might be extremely hot.
Autoclave:
When heating up bottles in the microwave ensure the cap is open to avoid overpressure and explosion. Wear gloves when taking the item out as it might be extremely hot. When opening the lid make sure not to put you head just above as some exhaust steam might hurt you.

Storage:
The lab contains several storage spaces. A fridge (F), a freezer (F+), a large vertical cupboard (S1) and a few bench cupboards. Each storage space should be organized in a precise way, to only contain the material aforementioned (noted on the shelf itself).
to re-organise still !!

Legal:

According to swiss federal law, before starting an experiment which may be classified as containing class 1 organisms, the following procedure should be followed before and after the experiment is performed.

First a thorough risk assessment should be performed on the microorganism that is being worked with . Use these resources to evaluate the organisms and control these points (list is not exhaustive, full list found here).
Pathogenicity and lethality
Virulence
Route of infection, infectious dose
Production of toxins or potential allergens or oncogenic peptides
Reproduction and survival mechanisms
Potential hosts
Immunity to treatment/chance of acquiring it, resistance to antibiotics and other treatments
Existence of treatments against the organism
Mutagenicity
History of other laboratories working with the same organism
Measures should be taken to minimise risks
Produce a detailed record of what you are about to do (continue doing so during the experiment).
Decide whether to notify the authorities.
Notify the authorities about any changes while the experiment is being performed.
After completion of the project, update the status of the experiment.