- Hi everyone, I'm Doctor Lentz
and today I'm gonna guide you through
the process of aseptic technique use
in the bio safety cabinet and more broadly,
in cell culture work.
Now you'll notice we're starting out,
I'm already gowned up in personal protective equipment,
my (mumbling).
This includes my gloves, my lab coat
as well as eye protection.
We also are going to be working in
a bio safety cabinet that has already been sterilized
so I'm prepared according to the techniques
that you would have learned in the previous video
relating to that.
Alright, in aseptic technique, the purpose
is to work with biological agents
that we need to preserve the sterility of.
The goal being, we want those biological agents to grow,
perhaps a cell line or cell culture,
but we want to prevent contamination from bacteria,
fungi or other biological agents
that we aren't interested in
or that might affect or impact our cultures.
Today, we're going to be working through
the process of splitting cells.
For a first step, we need to prepare the materials
we're going to use in that process
and that includes the media that we're going to add on
to the cells, as well as the cells themselves.
Now the media, I got ready to go right here.
We're going to now go and collect our cells.
So the cells will be stored in an incubator in the room.
Depending on the room's space, your instructor
will direct you to the correct incubator.
Different cell lines can be maintained
in different conditions and in different medias.
We aren't going to discuss today or in this video
the type of cell lines that you might work with
or the medias that you might encounter,
but your instructor should direct you
to this material, this information.
In this case, we'll be working with cells
that are present on a 10 centimeter dish.
They won't be visible in this video,
but you'll have to trust that they are present.
Over the cell line, I'm gonna set them
into the already prepared hood
in order to minimize their exposure to the environment.
Now, I'm not going to immediately
place the media tube in the hood
and that's for a specific reason.
Certain materials that we move into the hood
we need to sterilize before they can be used.
This includes plastic, hard plastic tubes and containers,
such as our media bottles, our PBS (mumbling) and bottles
that you might be using.
Other materials, such as pipe headers or pipette aides.
The cell culture we are not going to treat with ethanol
because in treating or spraying down that container
we risk contaminating or introducing ethanol
into our cultures, which could be equally damaging
as contamination itself.
However, we are going to spray down the media.
The spray process is pretty generic.
Your goal is to cover the surface of the item.
And (mumbling) that can be accomplished with a few spray.
I'm also going to transport, in this case,
the appropriate rack for this tube.
And then spray down that rack as well.
In this case, either of those items
could have carried in a potential contaminate.
Now we placed the media and the cell cultures in the hood
and these are the only agents we're going to need
for the procedure we'll be performing today.
In the case of your particular lab,
the instructor may direct you to include more materials
or you made need to introduce more materials into the hood.
In any of those processes, and in all of those processes,
to preserve aseptic conditions, you want to have those
materials ready in advance and move them all into the hood
at one point in time, similar to what we've done
with our tube and the tube rack.
You want to spray them all down with ethanol
and in doing so, you've now prepared a clean container
in which you can work with your cells.
As we work with our cells, we want to be conscientious
that we don't move in and out of the hood too often.
We're performing this work in a bio safety cabinet
and the way the bio safety cabinet functions,
the way it preserves an aseptic environment
is it filters the air in the top,
air passes through a type of filter,
and then passes that air in two sheets.
One sheet, or column, going into a grate on the front
of the hood and a second sheet of air
passing along the back wall of the hood
and entering a grate in the back of the hood.
Both of those sheets of air act as walls,
preventing any material from floating
or dripping into the sterile space within the hood.
We now have those conditions present.
So I'm gonna walk you through changing the media
on a cell culture plate in this aseptic environment.
Now once inside the hood, you would keep your hands steady
and you don't want to move them in and out.
We have our media, our cell culture plate
and we also need a few items present in the hood
in order to successfully change the media.
We need to have an aspirator, which we will use
to draw off the media already present on the plate.
We need pasteur pipette tips,
which we will insert into the aspirator
and this will be the sterile instrument
that actually encounters the cell culture media.
We need a bleach solution
that we would use to sterilize
the pipette tip once we finished aspirating.
And we also need to be sure that we have a biohazard bag
in case we generate any waste that we aren't intending
in this process.
Alright, the first step in splitting our cell cultures
is to turn on the aspirator.
And we should in doing so now have a suction
that we'll use to draw up the media in our cells.
We have our pipette tips ready
and we have our media ready to pipette on.
The final step before actually changing the media
is to make sure we have the pipettes to replace
the new media onto the cells.
For that, we're going to use a pipette aide
as well as pipette tips.
Now in grabbing or acquiring a pipette tip,
this is the one instance in which you'll probably find
yourself leaving the hood,
grabbing the appropriate pipette tip
and resuming work in the hood.
Try to minimize the frequency of grabbing pipette tips,
but that probably will be an item that you need
to acquire throughout the process.
I'm going to begin
splitting the cells by first opening
my pasteur pipette to have it ready to go.
I'll then take the aspirator,
place a new aspirator tip,
this now being a sterile instrument.
I'll then briefly remove the top of the plate
exposing the culture media and with one hand
steadily tilt the media so that it collects
on one side of the plate.
I then introduce the pipette,
which draws the media up.
Remove the pipette, quickly replace the lid.
Treat the pasteur pipette tip with bleach
and now remove this tip
and set it to the side for disposal.
This process, in this process, we need to be timely.
So I will quickly acquire my pipette,
place it into the pipette aide.
Replace the media.
In this case, I'm using a volume of approximately
eight mils.
I'll then briefly remove the lid of the plate,
holding it steady with one hand,
provide the media to the wall of the container slowly
so that the media does not rush in and disrupt
the cell culture.
Then quickly replacing the lid of the plate.
I then, with the used pipette re-wrap it
in the container it came out of
and remove that from the pipette aide
and set it to the side for disposal.
The pipette aide, I can return to its rack.
A few points that I'd like to highlight
in this process that are important
for aseptic technique, any time you move in and out
of your plate, you want to be very brief in removing
the lid of the container of the cells
and you want to handle it with a steady grip
so that you don't lose control of that plate
and perhaps spill some media.
Once the media has been changed,
our cell cultures can now be carried back to the incubator
and we can, afterwards, return and clean the hood
for the next user.
In our final step of preserving an aseptic condition
in our hood, we're going to spray down the surface
on which we were working with decon.
This is a cleaning solution meant for inactivation
of biological agents.
You'll find spray bottles of decon
that look very similar to ethanol,
distributed throughout the lab space.
Spray a thin layer of decon solution
on the surface at which you worked.
And wipe that surface with a paper towel.
In wiping up the decon solution,
we've now terminated or destroyed
any biological agents on the surface
in which we were working
and we won't leave any decon residue on other surfaces.
Lastly, we want to spray down the hood again
with ethanol and wipe up the ethanol.
The purpose of this is to help remove
any residual of decon agent as well as
provide one last decontamination step
prior to closing the hood.
The paper towel that you've used to clean up the hood
should be disposed of in the bio-hazardous waste.
And you have now completed the process.
You can close the hood.
Turn off the lights and the blower
for the next user.