This content outlines the essential facilities, equipment, and procedures required for establishing and maintaining a plant tissue culture (PTC) laboratory, emphasizing sterility and controlled environmental conditions.
Mind Map
Click to expand
Click to explore the full interactive mind map • Zoom, pan, and navigate
culture techniques and their general requirements
for a plant tissue culture.
The basic facility that should be available
available
usually include a washing and storing
area, media preparation, sterilization
and storage room, transfer area for
aseptic manipulation,
culture room or incubators for
maintaining the cultures in a controlled environment.
environment.
Then finally observation or data
collection area. So whether your lab is
a big lab or a small lab these areas
okay should and must be compulsory be there.
So coming to the glassware washing area
and storage room. The main requirement
for washing and storage is an area
sufficient to accommodate large sinks.
Okay. And this sink should be coated
with or lined with lead.
Why? Because it should resist the acids
and alkalies which we are pouring
sometimes into it.
And there should be provision for hot
and cold running water. There should be
a draining boats or racks
wherein you can dry your glass.
There should be provision for distilled
water and demineralized water
and area or space for keeping your
washing machines and ovens.
There should be plastic and steel
buckets for soaking your lab.
And once you wash, you can dry your
glassware by air drying it or you can
put it into an oven. So that provision
should be there, a place should be
there, provided there. And once you have
washed, you should put all your glass
into a dustfree cupboards or storage cabinet.
cabinet.
We shall see that how we can wash our glass.
glass.
Now most of you know that whenever we we
are in tissue culture lab we
particularly or any lab for that matter
we are using lab ve which are
particularly glass ve. So when you're
washing glass ve usually you have to
soak it in 4 hours in sodium dromate
and sulfuric acid.
Then wash it vigorously with the tap water.
water.
And then you wash it again with
distilled water. But you should take all
the precaution because you're using
corrosive solvents here like
concentrated sulfuric acid, sodium
dicromate which can be harmful to your
eyes and skin. So you should take all
the precautions. Otherwise you can avoid
this method sodium dicchloride and
sulfuric acid method. You can use detergents.
detergents.
Okay. The glassware is soaked in a
detergent solution for 16 hours then
rinsed first with tap water then rinse
it with distilled water. So kindly
remember that whenever your washing is
done you should wash it all your
detergents or whatever like chromic acid
you're using it should be washed again
with tap water and finally distilled
So suppose say you have dishwashers in
your lab that time how can we wash.
So the glass may be treated with
detergent due to detergent at 70°
centigrade for 5 to 10 minutes. Further
cleansing it involves 5 minutes rinse in
hot water 3 minutes in deionized water
and then finally you again have to wash
it with distilled water.
There may be some problems when you
encounter like washing the glass
particularly glass ve containing agar.
So agar would have dried on the glass ve
so it is not possible for you all to
So how can we remove this? Then the
glass wver there is a dry agar first you
have to remove the agar by melting at a
low temperature and then you wash it
with detergents and perform the same
steps as you performed in the others.
See preferably whatever glass we are
using it should be borosyicate glasses
is preferred. Why means it is less prone
to breakage or even scratching. So there
are very good companies like Pyrex,
Corning wherein they supply very good
glass which we can use it for our uh
tissue culture. So I'll be uh I'll be
not be mentioning tissue culture always.
I'll be mentioning the word PTC plant
Now we know that these glass bars are
prone to breakage. Okay. So instead of
using glass BS nowadays what we prefer
is the plastic w so we can use pre-sterilized
pre-sterilized
already sterilized polyester or reusable
plastic w to tell is like when you're
purchasing itself the glasses are pre-sterilized
pre-sterilized
okay you can just use them and you can
dispose them discard them but again what
happens this becomes a
very costly procedure for us because we
are buying and then throwing off. So now
what we have is we have reusable glass W
sorry plastic WS and these plastic WS
are particularly polyethylene acrylic
polyine. So if you want to wash this
plastic wear, you should have a
knowledge of what type of plastic wire
I'm using whether it is polyethylene or
polyerine because based on your glass
plastic w only you can wash your all
this plastic wires
I'm telling particularly reusable ones
because disposable ones you use and you
can throw but reusable again you can go
for washing. So you have to take certain
measure by washing the plastic bag like
rinsing with organic solvents such as
alcohol okay or acetone chloroform is
not practice for acrylic plastic bag.
You should not use these solvents when
you're using acrylic plastic B.
Similarly, like soaking in one normal
HCl for 8 hours or less followed by
another soak in one normal uh nitric
acid and rinsing in in water. This is
most acceptable form.
Okay. Then uh you can soak it in crobic
acid for less than 4 hours or mild
detergent you can use. So this also can
be used and also boiling it in dilute
sodium bicarbonate. This treatment is
generally recommended for polycarbonate,
polyethylene and acrylic or polyester
only those can be used. When you're
using dilute sodium bicarbonate these
all plastic W whatever we have mentioned
here we can use so we should be careful
like what type of plastic B we are using
whether it can withstand that tolerance
of that solvent or acid or detergent
whatever we are using.
Now this is a pictorial diagram of a
typical plant tissue culture lab.
So here we see that the next point is
media preparation room. How should our
So you can see here
there first there should be an ample
space for storage of your chemicals,
culture vessels
or any other lab wear or the
miscellaneous equipments which you want
to use for media preparation and
dispensing them.
There should be provision for placing
hot plates, steros, pH meters, balances, semicrobalances,
semicrobalances,
water bath, buns and burners. These are
all in equipments which are or
instruments required for your media
preparation. So that that is why there
should be an ample space to provide all
the spaces for these instrument
simultaneously like you are will be
doing the procedures for whatever you
want to do. So there should be proper
place for you also to do all this procedures.
procedures.
Now suppose say your lab is small. If
the preparation of the media and the
washing of labar are done in the same
room, it should not be done because
there will occur contamination. And we
have told that the biggest culprit of
our tissue culture is always the
contamination. But suppose say you have
a washing area and the media preparation
room in the same area then a temporary
partition should be constructed between
these two so that there is no any interference.
Also there should be supply of water
because we use distilled water for
preparing all this media and even for
washing our lab. So we should see that
there is a proper supply of continuous
supply of water.
See these are the some of the
instruments which I mentioned. See this
is a spectrophotometer
usually which we use vortex for mixing
your sample. Water bath balance okay
centrifuge tube laminina hood micro oven
incubator ster bunen burner
refrigerators autoclave. So these are
the common instruments or equipments
which we use in our PTC lab. So there
should be provision and place to
maintain these all in the media
preparation room because you can't run
about there and here for each instrument.
So this picture you can see a typical uh
tissue culture uh lab. It is a small lab
and uh this is the rack wherein you can
Again coming to your media preparation
area the benches suitable for
comfortable working. Okay the whoever
the person is working there he should
feel comfortable while standing and
working there. So the benches should be
at least 3 to 3.5 ft
and long enough about 6 ft. Okay. So it
should be 3 to 3.5 ft and at least 6 ft
so that the person can comfortably use
his space to do his work
and it is also ideal to have one or two
Coming to a transfer area.
Okay, the third point what we were
speaking that is a transfer area. Now
what do you mean by transfer area is? So
a transfer area is used for aseptic
manipulation. If I want to do any
manipulation in my culture, I cannot do
it in openly. I should come to a
transfer area and there I should do my
all my um manipulation under aseptic conditions.
So like we'll see that what is required
You can see the picture here. The
simplest type of transfer area is an
enclosed plastic box or a glove box
which can be sterilized with an
ultraviolet light. So here there will be
an ultraviolet light. So you can
sterilize this area. But remember
whenever UV light is on no individual
should be present there.
And then you can swab the floor okay the
surface with 95%
ethile alcohol.
Ordinarily a small wooden hood may also
be used for tissue culture work. The
hood has a plastic or a glass door
either sliding. It can slide or it can
be hinged removed up. Okay. Like this.
So the door should be dust proof. The
culture hood can be conveniently placed
on any benches because it is very small.
Okay. And whenever an individual should
do any manipulation, he can insert his
hands into this holes which is
containing the gloves and he can do all
the manipulation by seeing through the
Then after the work is done again you
can switch on the UV light or before
that you can swab your area and then
switch on your UV light so that again
the sterilized condition is maintained.
So this is a simple
uh equipment wherein you can keep it in
inside your tissue culture lab whereever
convenient you feel. So
so one of the drawback of this glove box
is whenever you are doing okay it has
very limited application because only
few transfers can be made here. When you
want to do large transfers there is no
sufficient place to do here. Okay. So
when large number of culture or transfer
are being man you have to do or do any
manipulation large equipment is required
and the most desirable is a dustfree
room. Okay a room should be available
and that room should be dustfree and
that room should have a UV light.
Okay. So the same procedure you have to
switch on your UV light. At the same
time that room should also have a power
ventilation unit.
So this ventilation should possess a
high efficiency particulate filter.
Okay. That room should contain what? It
should contain heapa.
Okay.
Okay. High efficiency particulate air.
So this is a filter and that filter is
fixed in the room wherever we are
performing our experiment.
So it has been observed that
0.3 micrometer HEPA filter show. Okay.
The filters the size is only 0.3 0.3
0.3 micrometers
micrometers
and it is shown that it can remove at
least 99.9%
okay 99.9.7
or 9.8% of microorganisms. Look at its
So here also you have to maintain all
your surfaces clean. Apart from this,
this is I'm speaking about the room.
Okay. Where in a a small room you can
use it with having UV light, having hea
filter and also a positive ventilation,
pressure ventilation. Otherwise what you
can do is we can go for the laminina cabinet.
cabinet.
Okay, this is the laminar cabinet. This
is also called as l
So lamina air flow.
We shall see about that. So
So
see this is your
laminina airflow cabinet. Okay. It is
written here.
So here
what we do is in this lamina air cabinet
a small motor. Okay see here this picture
picture
motor blows the air into the unit
through the coarse filter. So the air
passes through the there is a filter
which is a coarse filter and what is the
application of this course filter is it
tries to remove okay all large dust particles.
And then subsequently then it passes
into the hea filter here. The same hea
filter which is having 0.3 micrometer
filter size which can filter off all
your microorganisms.
So this is another advantages. So the
air now when you open this the air will
be flowing. Okay. Because you have
switched on the air blower. The air is
directed either downwards. Okay. The air
come can come in this direction downward
or it can come under the horizontal
direction facing the uh person who is
sitting here.
So particularly in plant tissue culture
what they do is the horizontal way of
air flow will be there. So we need not
be panicked that it will be because we
would have kept a bunen burner and all
here or spirit lamp. So there is no need
to panic because this will not the air
will not hamper our bones and burner any
of the flames. We can proceed with the
experiment. So this is the
uh pictorial diagram of laminar and this
is how it appears. So, so even this has
a glass or plastic particularly plastic
windows you can open it and you can
perform after swabbing with alcohol your
floor then you can perform all your
manipulations. So first switch on your
UV light.
After the UV light should be on for 10
to 15 minutes at least. And then you can
switch off. Then switch on your
illumination light.
Okay, the white light. And then swab
your roll with the 95% alcohol and
perform all your experiment. After your
experiment is done, again you clean the
area. Okay? Then close the door. Switch
off your air blower and then switch on
your UV light. After 15 to 20 minutes
So this is what we spoke about. It'll
have a small motor air flow through a
cross filter wherein it can remove the
The next one,
the last area what we require is the
culture room. Now what is the meaning?
Why do we require a culture room?
Culture room is required to maintain the
culture under controlled condition
and proper temperature, light and
humidity. So whatever cultures you have
prepared, you have to put them under the
culture room. a separate room so that
your plant can grow under the control
condition. So you have to provide a
proper temperature, light, humidity,
whatever the external environment,
natural environment is pro providing for
the plant that is you are providing into
your um tissue culture. So they are most
pampered once they looked after it
carefully. So what is the temperature we
have to maintain? The temperature water
we have to maintain is 25° centigrade
plus or minus 2°.
So we can maintain the control air
conditioners and heaters. So it should
like uh light and the temperature should
be programmed for 24 hours and the
lighting is adjusted in terms of
quantity and photo period duration that
is they should be having an automatic
clock so that you can provide 16 hours
of day and then the clock can click
telling you that okay 16 hours of
daylight is over then what you can do is
then 8 hours of night you can keep your
plants under the darkness. So what is
happening we are providing the photo
period we are imitating the external
environment the day and light. So this
is called as a photo period. So we keep
an automatic time clock to inform us
and also see this is a typical uh
So you can see here
you we have this iron uh cabinets or
racks. These racks the floor of this
racks can be made about glass or wire
mesh or even plastics. So whatever the
cultures are present
inside my glass ve I can place my glass
here. Okay, this is a temperature light
and humidity control room. So I can keep
that there will be no contamination. It
is a very highly sterile area. But
suppose say my culture is in the test
tubes. Then what should I do? I should
keep it in the racks otherwise they will
fall. So like 24 uh holes rack that one
we can keep it. So if you see here there
will be light here beneath each rack
there will be illumination light. Now
what happens this light is going to
generate heat and this can affect our
besides whatever racks are there. So
what to do is we can insulate the light
so that the air or the light flows
uniformly. Okay, the air and the light
flows uniformly.
Otherwise what you can do is if you want
for air flow to flow to the individual
racks then what you can do is you can
fix a small fan
and the air from that fan no it can go
into the pipes which are fixed here and
these pipes will be having holes.
These holes are should be equidistantly
maintained and from the heat of this
Okay, that is what here insulation
between the shelf lines and the shelf
above will ensure an even temperature
around the cultures what we have
maintained. We can keep the flask. Petra
dishes can be placed directly on the
shelves or tray.
But whereas for the test tubes you
require some sort of support that is the
polyropylene racks which we use which
will have a capacity to hold about 18 to
So here also we have an alarm system. It
to indicate when the temperature has
reached whatever the temperature limit
whether it is going above the limit or
below the limit that will be indicated
by an alarm system which is fixed and we
have also have a temperature recorder
which will take because we simply cannot
go into that room each time to monitor.
So what happens is we have this alarm
system and temperature control recorder
which tells us whether the temperature
is properly maintained like 25° means is
it maintained at properly or is it gone
above or it has gone below. Is there any
fluctuation in temperature is all
dictated by a temperature recorder. What
should be the humidity range? The
humidity range should be around 20 to 98°
98°
uh sorry 98%.
And the fluorescent light what we told
the photo period of light it should be
1,000 lux. So as I told your light and
temperature should be programmable for
24 hours. That is why you are keeping
all this alarm recorder everything so
that it sets up the things properly and
whatever you're doing the experiments
so suppose say I have a tissue culture
in some uh glassware I should label them
properly telling that what is the date I
have conducted the experiment what is
the type of culture I have used is it an
ovary culture or is it a leaf culture
I'm doing
and what I'm expecting when should I
come next and view all this okay this
all should be written down in the form
of label this is because like one person
has done the experiment and this duty is
over and the next person should come and
see that what the other person has done
so there should be a record maintained
So that the second person whoever comes
will not be blindfolded.
Immediately when he comes and does sees
the record the tag and the label okay he
should understand that everything
whatever the previous person has done he
So these are some of the instruments
which you require like pH meter, ster,
microbalance, autoclave which are
commonly used in the PTC lab and also we
require a microscope to visualize our
cells. What is happening to the cells
whether the division is occurring? If
the division does not occur if there
will not be any use in it. So this all
should be maintained properly. these
equipments or instrument.
Lastly coming to the observation and
data collection.
So here what happens is the growth and
development of tissue culture. We are
doing everything in in vitro. We have
taken what expplant what is the meaning
of the expplant? It is any part of the
plant. It can as I told you it can be a
whatever this ex cali or cells or what
protolast or the leaf or the buds
whatever you're using okay in vitro
you're doing they should be generally
monitored by observing the culture at
regular intervals in culture room or
incubator sometimes you have to keep
your cultures in the incubators. So
these should be monitored properly where
they have to be maintained under
controlled environmental condition.
Data based on observation and aseptic
condition may be collected using the
laminina airflow because you cannot open
your culture vessels inside your trans u
culture room and then you cannot do any
manipulation. Anything you want to do,
you have to bring it into the lamina
group where there is a sterile
condition. Okay, that is ultra sterile.
So there you have to come and do the manipulations
manipulations
and uh
also like uh you have the microscope
what I tell you told you for examination
of the culture. So a microscope should
also be maintained.
um we have to observe like when the
plant has started to propagate okay it
started to develop the plants
regenerated from invitro tissue cultures
should be transported or trans sorry
transplanted into soil in pots. Okay. So
you have pots wherein you put a sterile soil.
soil.
Okay. The soil should be sterile.
Okay, sterile soil you put and then
whatever the tissue culture plant is
there you put it plant it into this
sterile soiled plant.
Then the potted plants are ultimately
they are transferred into
a greenhouse. Usually this greenhouse
may be a glass or sometime even the wire
meshes are also used to construct an
greenhouse and there you can look after
the plant like again there you have to
provide all the control conditions of
light, temperature and humidity. You
have to see that whether the plant is
stable enough, whether it can withstand
the external environment, whether it is
ready for field to go to the field. And
then when you are give a thumbs up that
okay this is ready, this plant can go to
the field then you have to shift the
from the plant from the greenhouse into the
the
uh tree. Uh this is lastly you can see
that uh this is an shaker. Whatever
shaker I told okay it they keep it into
the tissue culture suppose say we'll be
studying about suspension culture and
all that time the shakers are very
important okay see how they have kept
the media is there okay it is plugged
with cotton and then a brown paper is
wrapped also and some or it is and then
you can switch on it and it'll be
shaking okay your material inside it
will be shaking
so these are some of The
instruments like forceps, scarcals all
which are required which then we'll deal
these all the instruments what is
Click on any text or timestamp to jump to that moment in the video
Share:
Most transcripts ready in under 5 seconds
One-Click Copy125+ LanguagesSearch ContentJump to Timestamps
Paste YouTube URL
Enter any YouTube video link to get the full transcript
Transcript Extraction Form
Most transcripts ready in under 5 seconds
Get Our Chrome Extension
Get transcripts instantly without leaving YouTube. Install our Chrome extension for one-click access to any video's transcript directly on the watch page.
