Tissue Culture Room

Tips for a Happily Functioning Tissue Culture Room

You walk into your tissue culture room to find a window open, the incubator’s humidity tray empty and a pipette lying on its side in the hood on a used glove

After you have found and torn to shreds the person responsible for this monstrous act (!!), consider posting the below tips to help those new to tissue culture or those in need of a refresher remember some essentials!!

Tissue Culture Room Checklist

After Entering the Tissue Culture Room

  1. Put on a lab coat and gloves. Ideally, lab coats should live in the room and never be removed.
  2. Ensure there are no open doors or windows. Drafts bring in bugs and disrupt your hood’s airflow. Reduce foot traffic into the room as much as possible.
  3. Turn on the hood and leave it on for at least 20 minutes before using it. Spray with 70% ethanol.
  4. If you will need it, turn on the water bath. Be sure to set the temperature and keep an eye on it. Use a thermometer to check it isn’t too hot or cold. Add your media, solutions, etc. when the temperature is stable.
  5. Wipe down all bench surfaces, microscopes, hemocytometers, etc. you plan to use with 70% ethanol.
  6. Think about what you need to do in the room and plan so that you move in and out of both the hood and the room as little as possible.

The Incubator

  1. Check incubator temperatures, CO2 levels and the humidifying tray daily (especially before the weekend). Add sterilized water to the tray if necessary.
  2. Check all your cells daily to ensure infections are caught early on.
  3. Spray anything that leaves or enters the incubators with 70% ethanol.

The Hood

  1. Wipe the working surface of the laminar flow hood with 70% ethanol.
  2. If using a vacuum, check the pump is functioning and that the aspirator is clean and contains Clorox or a similar bleach solution.
  3. Wipe any media bottles, etc., with 70% ethanol before placing in hood and after removing them from the hood to avoid putting contaminated bottles in the incubator.
  4. Do not use Bunsen burners in hoods as the flame disrupts the airflow.
  5. After using the hood,wipe the interior with 70% ethanol, turn off the vacuum pump and discard any waste into suitable bags for autoclaving. Check that there is sufficient Clorox in waste flask and the flask is not over-full.
  6. Clean anything that lives in the hood regularly, especially the pipettes and any tubing.

When Leaving the Tissue Culture Room

  1. Check you’ve put everything away neatly.
  2. Close and turn off the hood if the next scheduled person to use it won’t be there for 2+ hours.
  3. At the end of the day remember to switch off microscopes, turn off water baths, hoods, etc. and check to make sure incubator doors are closed, and lids on liquid nitrogen tanks are replaced properly.
  4. Empty the bins, top up anything that has run out, and leave the UV light on for at least an hour (with the hood closed.)

Maintaining the Tissue Culture Room

  1. Thoroughly clean water baths regularly and replace with fresh, clean water.
  2. Leave hood equipment in its place. This ensures they are as clean as possible. Keep tips, Falcons, pipettes (supported vertically) neatly on one side. This is the sterile side. Anything contaminated (e.g. used tips, containers) should be kept on the other side and removed as quickly as possible. It is better to keep it in the hood until you are finished working to avoid taking your hands in and out to a bin and risking contamination.
  3. Observe cell cultures microscopically daily to become familiar with the morphological appearance of different cells. This will lead to an ability to see when irregularities and contamination occur which, in turn, may allow one to rescue or discard a culture at an early stage.
  4. Do not keep cell stocks in culture for more than three months. Recover a new vial and make sure cells are tested regularly for Mycoplasma.
  5. Especially important tip: this tip applies for all electrical equipment that needs to stay on 24/7/365. Check that the device (incubator, -80°C freezer, fridge) is plugged into an outlet that, if the power were to be turned off, would remain on. There are special plugs for this purpose and usually they’re a different color.

Share your tips below!

4 Comments

  1. Bitesize Bio on October 16, 2015 at 10:59 am

    […] first thing you learn about culturing cells is proper aseptic technique and avoiding contamination. After that you’ll learn all the ins and outs of culturing your […]

  2. Olwen Reina on February 16, 2015 at 6:36 am

    Hi andreasb! Thank you for your ideas! Persoanally, I’ve always used either a strong detergent or bleach. What do you like to use?

    Hello AliciaHenn! That’s a very good point. If you use a diluted alcohol it tends stay longer but I would recommend not wiping it off immediately as people tend to do. You can actually let the alcohol sit on it overnight. I’ve not used the disinfectants you’ve suggested- thank you for sharing your wisdom :).

  3. andreasb on November 3, 2014 at 7:57 pm

    Well nice summary for beginners but Two very Important aspects are missing.

    1. you schould recommend a desinfectent for the incubator water. I Never had Problems with Cells using them. There are several commercialy available Products.

    2. mycoplasms can’t bee Seen in light Microscops so you should do from Time to Time a mycoplasms test. I Premiere the PCR test but also other Techniques are available.

  4. AliciaHenn on October 30, 2014 at 7:29 pm

    Just be aware that although the effects of alcohol at the pub can linger, that alcohol sprayed on surfaces evaporates so quickly that it does not disinfect well. You should not think of 70% alcohol-sprayed surfaces as disinfected. Try a more robust disinfectant like Vesphene or SporKlenz for better risk reduction.

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