Laboratory Fundamentals in Biological Engineering

Guidelines for Working in the Tissue Culture Facility

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Good cell culture technique will simultaneously protect you from anything dangerous that might be living with the cells and protect the cells from contamination by you. You will be working with an established cell line unlikely to carry any agents that could harm you. Consequently, the guidelines here emphasize techniques for maintaining healthy and uncontaminated cells. Some points are particular to the 20.109 cell culture facility but most are common practice and will be good habits for any tissue culture work you do.

Contents

• Maintaining cultured cells
  • Feeding
  • Splitting
  • Freezing
• Hood preparation
• Sterile handling
• Cleaning up

Maintaining Cultured Cells

Feeding

As cells grow and divide in a dish, they use up the nutrients provided by the media. Old media must be removed and the cells must be “fed” with some fresh media. This must be done every two or three days for most animal cell lines.

Splitting

Cells growing in a dish begin to crowd each other and then stop growing. This crowded state is called “confluence” and to maintain cells, confluent cultures must be “split” and “reseeded” into new culture dishes at a lower density.

Freezing

Every lab that works with cultured cells has a freezer stock of each cell line they study. The freezer stock is a critically important resource for the lab, storing lines that aren’t in use but are worth saving and also providing “back-up” cells if working cultures get contaminated.

Hood Preparation

1. Wear gloves to protect yourself but also to prevent dry skin and micro-organisms from contaminating your samples.
2. Use 70% ethanol for sterilization of non-sterile equipment and surfaces
   • Swab down the work surface liberally with 70% ethanol. Start from the back and proceed forward. Swab during work if necessary.
   • Swab any instruments that will be used in the hood with 70% ethanol, particularly the pipettes, which will often be used above biological samples.
   • Dry bottles thoroughly if they have been taken out of the water incubator. Swab them with 70% ethanol, especially at the neck and the bottom, and place them directly into the hood. Avoid shaking them vigorously during handling.
3. Open sterile equipment or culture dishes only inside the hood
   • Keep sterile pipette tips in “Hood Only” boxes that are opened only in a sterile environment. Swab the exterior of the box with 70% ethanol.
   • Bottles should always be tightly capped when outside the hood (i.e., they should have been tightly capped the last time they were in the hood).
4. 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.

Sterile Handling

1. Spray gloves with 70% ethanol as often as necessary, and/or change gloves frequently.
2. The indicator stripes on the autoclave tape should turn black if an object has been properly autoclaved.
3. Mop up any spills immediately and swab with 70% ethanol to prevent the growth of microorganisms.
4. Do not fill a dish/flask so full or swirl it such that the medium spills over the edge. This will introduce a path of infection via liquid and may cause cross-contamination.
5. Working with pipets
   • Withdraw a pipette from its wrapper at the center of the work area, tilt it so the tip (bottom end) is pointing away from the frontal non-sterile area and away from other objects in the hood.
   • Withdraw the pipette so that it slides through the sterile interior of the wrapper without touching the outside of the wrapper.
   • Handle the pipette with a steady hand. Avoid large motions and do not let the tip touch anything non-sterile. Keep the tip away from the front and far above the objects in the hood.
   • Avoid contact between the tip of the pipette and the mouth of the bottle. The mouth and neck of the bottle (both inside and out) present a potential source of contamination.
   • Never pour from one sterile container to another. Pouring will generate a liquid path to introduce infection from the outside to the inside. Always pipette or use filters when transferring from one bottle to another.
   • When working with Pasteur pipettes, do not reach into the box to remove it. Instead, shake the box gently to cause the pipettes to slide out slightly, and then withdraw a pipette without touching the other pipettes or the tube interior.
6. Minimize disruption of work area and around samples
   • Never block the negative pressure zone (also the frontal non-sterile area) of the vertical laminar flow hood with objects (i.e., notebooks, pipetteman handle).
   • Avoid working too close to the front of the hood. Keep working area at the center or towards the back. Keep the objects needed for the current procedure within reach; keep the others in the back.
   • Avoid working above an open bottle or dish in vertical laminar flow. Always work around them unless they are capped or covered.
   • To keep the hood from being cluttered, do not leave any trash in the hood. Discard uncontaminated wrappers in the regular trash. Put all pipette tips and biologically contaminated sharps in the sharps biohazard waste container. Put all biologically contaminated tissue culture plates, flasks, and other non-sharps in the non-sharps biohazard waste container (near the sink). Discard trash immediately or collect it in a small, containable pile that you periodically empty. The latter approach minimizes entry/exit from the hood in order to reduce disturbances in the laminar flow at the hood entrance, as these may create the potential to waft in contaminants.
   • Avoid leaving bottles, dishes, and flasks open when they are not in use. If the cap must be laid down, place it face-up/face-down towards the back of the hood where there is less traffic and less chance of being touched or crossed over. Correct cap placement has been debated. Having a cap facing up can potentially introduce airborne particles and drive non-sterile lid liquid onto the interior face of the cap, where contaminations can fall into the bottle upon recapping. If face-down placement is preferred, then make sure to swab the area specifically and thoroughly before the cap is placed down there. Conversely, if hood surface sterility cannot be absolutely guaranteed due to high traffic or cluttering, then face-up is a better option. The best placement, however, is to place the cap on its side and towards the back of the hood. This way the interior is not in contact with the air flow or with the work surface. However, this is not possible with dishes. Therefore, exercise good judgment in light of individual operating style and the hood setup.

Cleaning Up

1. Cap bottles tightly before removing them from the hood.
2. Swab down the work surface liberally with 70% ethanol.
3. Turn off the vacuum, if used.
   • Run a few mL of 10% bleach through the line.
   • Turn off the vacuum, and hang associated tubing outside the hood.
4. Close the hood.
   • These hoods operate best with the blowers on 24/7.
   • Do not turn the UV light on - to increase its lifetime, the lamp is used only sporadically.

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