Name: Sterile Cell Culture Technique
Uploaded: 2025-06-17T06:53:05.000Z
Duration: 13 min 22 s
Description: Thousands of YouTube videos with English-Chinese subtitles! Now you can learn to understand native speakers, expand your vocabulary, and improve your pronunciation...

This is a short demonstration of the techniques used to propagate animal cells.

The first step is to visually inspect your culture.

Remove it from the incubator and look at it.

The cells should be stuck to the surface of the culture vessel.

There should be no apparent mould or cloudiness, and the colour of the Put the flask onto the microscope platform.

Approximately 80% of the flask surface should be covered.

Also check that the cells are well grown, and that nothing is contaminating them.

This is a class 2 microbiological safety cabinet.

Air from outside the laboratory is drawn through the front and underneath the plates at the bottom.

It goes up a space in the back and is pushed down through the top through a big filter which removes dust and microorganisms so that the air which flows down to the working space is relatively clean.

To clean the cabinet we use 70% ethanol which is a very safe surface disinfectant.

Spray generously onto the cleaning paper and disinfect all the inside surfaces of the cabinet.

Make sure all of the surfaces are cleaned and ensure you work into the corners.

Once the inside is clean you begin preparing all of the equipment you are going to use.

Put on a pair of gloves and sterilise them by spraying generously with ethanol.

Make sure the entire surface of the gloves are covered.

The gloves protect your sample from the microorganisms on your skin.

Once your gloves are sterile, don't touch your hair or skin or you'll get contamination.

Sterilising all of the equipment using ethanol prior to placing it in the cabinet is one of the key ways of preventing contamination.

Arrange your equipment in the cabinet, to the right if you're right-handed.

Don't bring too many items into the cabinet as this can disrupt the airflow.

The purpose of cell culture is to remove the old culture media that the cell has been growing in and replace it with new media.

This is because the nutrients become exhausted and it also gives the cells more room to divide.

The first thing we need to do is remove the old media using a pipette.

The pipette is sterile so handle it using the plastic wrapper.

Load the pipette into a filler and remove the plastic sleeve.

Be careful that the pipette doesn't touch anything or you'll risk contamination.

Using one hand, remove the lid from the tissue culture vessel and place the pipette inside the liquid and use the top button of the pipette filler to suck the liquid up and then dispose of it into the disinfectant.

Replace the top as soon as you can to minimise the risk of contamination from the air.

Remove the pipette from the filler and dispose of it into the bin provided.

The next step is to add a buffer solution which doesn't have any nutrients.

Load a pipette into the filler, handling it with the plastic wrapper.

The buffer solution is used to wash off any residue of the old culture medium since the process that we use to detach the cells can be affected if there's any medium left.

Draw 10ml of buffer solution into the pipette and wash the buffer down the surface that the cells are stuck to, rinsing over the cells.

Draw the buffer solution back out and dispose of it in the disinfectant.

For health and safety reasons and to prevent any release of biological material into the environment, all biological material must be disposed of in disinfectant.

The next step uses a molecule called trypsin, a digestive enzyme that's found in all mammals.

It's used to disrupt the proteins which stick the cells to the plastic flask.

Again, use one hand to remove the cap from the trypsin and hold the cap in the same hand.

Put the trypsin on the surface of the cell culture vessel that the cells are growing on and replace the lid.

The trypsin is a digestive enzyme and works most effectively at 37 degrees centigrade.

Put the flask into an incubator for 3-4 minutes.

Don't leave the cells in the trypsin for too long or you'll remove the cells' surface signalling proteins.

After incubation, visually inspect the flask on the microscope.

Ideally, they should appear spherical but still be attached to the plastic.

If they're all floating free, they've been trypsinised too much.

Shock the flask by tapping to dislodge the cells.

Examine the cells again to ensure that they're floating free of the plastic.

Remember to sterilise the flask with ethanol before placing it back in the cabinet.

To arrest the trypsinisation and prevent damage to the cells, we add fresh culture medium which provides lots of protein to mop up the trypsin.

Take a pipette and open the container using one hand.

Fill the pipette and pour the fresh medium down the back wall of the flask, which washes the cells to the bottom.

Using the same pipette, remove all of the liquid from the flask.

And using one hand, remove the cap of a centrifuge tube and put the liquid in before replacing the cap.

Make sure that there's a counterweight before shutting the lid.

Spin the sample at 1800rpm for 4 minutes.

Be careful not to overspin or the cells will be damaged.

Take the sample from the centrifuge and inspect it to ensure that the cells are in a pellet at the bottom.

Sterilise it using ethanol before returning it to the cabinet.

Next, prepare a new flask of culture medium for the cells to grow in.

Take a new flask and load a pipette into the filler.

Fill the pipette with fresh media and transfer it to the flask.

Remove the lid with one hand and replace it as soon as possible to reduce the risk of contamination.

The next step is to re-suspend the cells using some fresh culture medium.

Invert the flask over the disinfectant in one fluid motion to remove all the culture media and avoid dislodging the pellet.

Then check that the pellet is still there.

Take a fresh pipette and attach it to the filler.

Flush the liquid in and out of the pipette until the pellet is broken up and is a single cell suspension again.

To avoid an uneven cell culture, repeat the process until there are no lumps floating around in it.

Take approximately 30% of the cell suspension and place it in the culture medium, then tighten the cap.

Examine the culture using the microscope to make sure the cell density is high enough.

Put the new cell culture into the incubator at 37 degrees.

All liquids go into the disinfectant for at least 2 hours prior to disposal.

The trypsin and fresh culture medium are replaced in the fridge.

All inside surfaces of the cabinet are cleaned and ready for the next user.

Lastly, any general waste and plasticware that is contaminated with biological material is disposed of in the biohazard waste bin.