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Contents

Lab safety

General Lab Rules

  1. Keep your bench as clean and organize as possible.
  2. Label everything. Keep track of all the solutions you prepare. Labeling basics are: Name, Initials of owner, date of preparation, concentration when possible.
  3. Do not do anything that you are not completely sure about what you are doing. Ask for help.
  4. Always wear gloves, unless you are working with the flame.
  5. You generally do not need a lab coat. If you really need one, ask *David.
  6. When working with UV light (DNA gels...), avoid direct eye exposure. Put the orange googles before turning on the light.
  7. The lab do not generally uses EtBr for DNA staining, if you need to do so, label everything you use as EtBr positive and ask *David for storage. Avoid direct exposure of your skin and be very cautious.
  8. When managing sharp razors, broken glass or needles, dispose them in the special yellow container.
  9. When one of the containers is full, call waste management "620 012 703" (Ricardo)
  10. As you know, safety conditions here at the INC are not optimal (no eye shower, no safety shower...). At this point, it is very little what we can do to improve that, so please be extra careful when managing acids or corrosive chemicals. Use the hood in the chemical room, and prepare all your solutions there.

Tissue Culture

This protocol describes techniques to be used for tissue culture in the SysBio lab: These guidelines must be followed!- failure to do so can result in damage to your and the experiments of others. To protect the overall lab, you need first to be trained by a certified Tissue Culture Master before beginning work. Only a Tissue Culture Master can certify you as a Tissue Culture Master that can train others, so be nice to them. Tissue Culture Masters:

         -	Adriana Sanz	

Space: You will be assigned a space in a tissue culture incubator. You will also be assigned a space at the refrigerator (4 C) and at the freezer (-20 C). You must use the hoods and incubators only in the room you were assigned. If you need space in the Criofreezer (-80 C) container for archival of cell lines, contact David G. Miguez.

Safety: Although most cell lines are reasonably safe to work with, be aware that you are working with or around potentially dangerous materials To protect everyone’s safety, follow these guidelines:

  1. Label EVERYTHING. Minimal labeling includes your initials, the date, and the cell line nomenclature. For cell passages, it is usually best to include passage # and split ratio for your own information. At no time can you leave unlabeled materials in the lab when you are not in the lab.
  2. Anything that has touched cells should be bleached and left for 15 minutes before disposing it in the sink outside the tissue culture room. Media/serum that has not been used on cells can be disposed of in the sink.
  3. If you reach the limit of volume marked on the aspirator, you should proceed to bleach it and dispose it as explain above.
  4. The doors to the tissue culture rooms are to be kept shut at all times. When in the tissue culture room, you can wear a lab coat.
  5. ALWAYS wear gloves.
  6. Before entering the tissue culture room. Think of all the materials and Fungible that you plan to use, to avoid as much as possible entering and exiting on the tissue culture room.
  7. Glassware has to be disposed at the special yellow container.
  8. Plastic ware that has touched cells has to be disposed at the specific container.
  9. All the rest can go to the normal trash bin.

Aseptic Technique:

Contamination by microorganisms is a major problem in tissue culture. Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions and many other sources. Proper aseptic technique seeks to eliminate these contaminants. It is important to establish and maintain aseptic techniques particularly given the importance of cell culture to everyone’s experiments in the lab. Contamination can be minor and confined to one or two cultures, can spread among several cultures and infect a whole experiment, or can be widespread and wipe out your entire stock. Catastrophes can be minimized if :

  1. Cultures are checked carefully under a microscope,
  2. Cultures are maintained without antibiotics for at least part of the time to reveal cryptic contaminations,
  3. Reagents are checked for sterility,
  4. Bottles of media are not shared with other people,
  5. The standard of sterile technique is kept high at all times.

All materials that will come into direct contact with the culture must be sterile. Aseptic technique is a combination of procedures designed to reduce the probability of infection. Most cell culture work is done in a laminar flow hood.

General Rules:

  1. Wear gloves – but note that these are to protect you, and are not inherently clean!
  2. Keep the work surface clean (clean with 70% ethanol and keep the work area clear enough to allow work without reaching over an item and preventing inadvertent brushing of a sterile tip against another object.)
  3. Use sterile reagents and media and work to keep them that way (do not reuse tips, clean the outside of reagent bottles with 70% ethanol, autoclave or sterile filter as appropriate)
  4. Keep bottles, flasks and tubes covered as much as possible.

5) Remove liquid from containers at an angle.

6) Clean with 70% ethanol often (surfaces and gloves).

7) Remember that it is the inside of containers (autoclaved bottles, centrifuge tubes, etc) that is sterile.. treat the outside as dirty and clean with EtOH.

Start-up in the hood :

The major advantage of working in a laminar flow hood is that the working environment is protected from contamination by a constant flow of filtered air. A vertical flow hood is where air blows down from the top of the hood onto the work surface. Follow these guidelines when using the laminar flow hood. Keep this direction of flow in mind when setting up your work area.

  1. The hood must be operating correctly to be used- be sure UV light is OFF, turn on regular light if needed. Turn on the hood blowers before opening the hood cover (carefully not to damage the UV lamp). Hood blowers are not to be turned off- if they were turned off for any reason, run for 15-20 minutes to clean the air in the hood.
  2. Remember that everything that goes INTO the hood must be cleaned with 70% EtOH. This includes your hands. Spray them down before starting.
  3. Swab down the work surface liberally with 70% EtOH. Wipe off anything you intend to use in the hood (pipettor, vacuum line/valve, Pasteur pipet box, etc).
  4. Bring stuff into the hood that you intend to use – pipets, tips, etc. Each of these items needs to be cleaned with 70% EtOH. In addition, if you plan to use tips or microcentrifuge tubes, be sure to keep an autoclaved stock that is kept for hood use only.
  5. Dry media bottles thoroughly if they have been taken out of the water bath (this water is a great source of contamination). Swab with 70% EtOH, especially at the neck and bottom before placing in hood.

Working in the Hood:

  1. As stated above, everything that goes into the hood must be wiped with 70% EtOH before putting it onto the bench in the hood. This means your hands, new boxes of tips, new beakers of tubes, the exterior of bottle-top filter units, etc. Everything.
  2. Keep the air intake (the vent in the bottom of sash, underneath your elbows) clear of all items at all times. If this vent is blocked, it allows room air to enter the hood, which is great source of contamination! Also, keep items from directly blocking the vent in the back of the hood.
  3. Bring only the items you need for a particular procedure into the hood to prevent cluttering your working space. Having a clear working space will significantly reduce the chance of contamination! Ensure easy access to items in the hood and maintain plenty of clear space in the center of the hood to work in.
  4. This is a vertical laminar flow hood, which means that air flows straight down from the top of the hood. Do not work directly over any open vessels, or contaminants from your hands could be blown into your vessel. Always work at an angle, off to one side.
  5. Watch what you are pipetting! The replaceable filters in the air pipettors are expensive. Ensure that you don’t suck fluid up inside them. Especially watch the 1 mL pipettes...they fill really fast!
  6. If you spill anything in the hood, clean up immediately to prevent cross-contamination and damage to working surface. Stop what you are doing and wipe up the spill – salts in particular can corrode the metal if left. Wipe the area with 70% EtOH before returning to work.
  7. Styrofoam is not recommended in the hood – it often flakes and is difficult to keep out of the vents. Use plastic racks instead (if you need more racks, be sure to clean with EtOH before putting into the hood).

Using the UV Light:

Although not recommended for routine sterilization of the hood itself, each hood has a UV light which can be used to sterilize surfaces, etc.

  1. Put in surface to be sterilized. Turn off blower and shut sash.
  2. Turn on UV light for desired time (10-15min is usually sufficient).
  3. Turn off UV light, open sash to upper marked height and turn on blower.
  4. Let blower run for ~20 minutes and proceed with normal EtOH start-up.

Incubators and Microscope:

These are shared equipment, so they present a great method to spread contamination!

  1. Before using the microscope, spray a piece of paper with 70% EtOH and wipe down the stage. Do this also when you are done to prevent media spills from spreading between plates.
  2. The incubators are not technically sterile- however, every effort must be made to maintain their cleanliness to prevent contamination from spreading. If media has spilled in the incubator, clean the spill with EtOH. Spills into the water bath MUST be immediately taken care of- talk to either the lab manager or the tissue culture czars.
  3. Put plates carefully into incubator- be sure not to bump other people’s plates, minimize stacking as much as possible, and keep your own plates set up to allow you easy access to what you need next.

When You Are Finished

  1. Trash must be disposed of properly
  2. Remove all of your media/solution bottles (tightly capped), tubes, etc from the hood. You should leave inside the hood only what it was in it when you arrived.
  3. Be sure the pipet-aid is returned to its rack to turn off pump.
  4. Wipe down the bench with 70% EtOH.
  5. Put the cover back if no one will be using the hood immediately after you.
  6. Turn off the air and turn on the UV light.

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