Maintaining aseptic conditions and employing effective sterilization techniques are paramount in plant tissue culture (PTC) to prevent microbial contamination, which can lead to the loss of valuable plant material and its desired products.
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So another PTC today we shall see about
the aseptic conditions and
sterilization. This is
an important topic.
So what do you mean by aseptic condition
and why one should do?
This is because we are growing our
plants in vitro and we are providing all
the conditions the environmental
condition what is available outside to a
plant. Simultaneously we are providing
it with media which consists of
nutrients. So usually what happens is
the microorganisms
okay they also
try to invade the media and start
proliferating in it. So that is why we
have to see that there is aseptic or
completely sterile condition available
otherwise it'll be contaminated by your microorganism.
And one more point is always when there
is contamination by microorganism we
know that the microorganism generation
time is very fast. So they outgrow the
plant that is they can grow faster than
the plants and they can suppress the
growth of the plant
So that this is the reason why we should
follow the sterile condition or aseptic techniques
techniques
and also you you know that the
environment okay the which consists of
the air is fully highly contaminated.
For example you can see that sneezing
itself can produce 100,000 to 200 uh
thousand droplets of microorganism.
So you can just imagine how much
contamination it is causing to the
environmental air. So these contaminated
particles okay from where whatever the
people are sneezing or coughing whatever
it consists of many microorganisms
and apart from microorganisms it
consists of even the spores. So it is
very difficult to eliminate the spores
and the frequent contamination what we
see in the PTC is the bacterial contamination.
So usually what we do is whenever we are
using our expplant okay expplant as I
told you it is a small piece of plant
whether it is leaf bud or ovary or
anther anything. Okay, it is a part of
the plant is called as a expplant. So we
are using the expplant
to develop it into a fully grown plant
which means it that is the meaning of
totency. So the di are usually
introduced with the expplant who that is
the microorganisms.
So whenever you're taking the expplant
to grow it in the laboratory
you are sometimes adding even in your microorganisms.
microorganisms.
This is because always the expplant
whatever we take no it is always surface
sterilization we do only the surfaces of
the expplants are sterilized. But what
about the interior of the tissue? If the
microorganism is present in the interior
of the tissue and when you start
developing that expplant automatically
it gets contaminated because the
microorganism was hidden within. Okay,
you have just done the surface
sterilization but the hidden
microorganism within the interior tissue
escapes the sterilization. So this can
lead to contamination also.
also.
So to avoid contamination last class
itself we told that we are going to use
laminar airflow.
See this laminina airflow also can get contaminated.
So what we have to do is wherever
whenever you're placing laminina hood.
Okay that is the laugh. Whenever you're
placing the lamp you should see that the
laugh is not directly
towards the door.
Okay, because the doors you'll be simply
opening. So what if you place if this is
a door and if you're placing the
laminina hood here and frequently the
door is kept open life will get contaminated
contaminated
otherwise if there is no option at all
then you have to keep a double door.
So here the people can walk about you
can just open this door and then open
this door then you have the laugh.
So also the
individuals who are using who are doing
this plant culture even they should take
lot of precautions. Okay. Like they
should wear sterilized
uh cloth or head gale and before
entering into the sterilization area
they you they have to see that they have
swapped the hair hands with alcohol.
and uh also some other like you're not
supposed to talk, not supposed to
sneeze. So these all precautions you
should take. So cleanlinesses and proper
care of equipment are vital operations
of the tissue culture laboratory.
So besides this contamination, you
should see that all your equipment
whatever you are operating even they are
under the sterile conditions.
So usually what
a tissue culture individual I told you
he himself should be sterilized
wear all sterilized clothing
clothing
and also he should see that he
sterilizes the hand frequently I told
you that using at least uh 75% of alcohol
So whenever we are sterilizing you can
see that if the spores are present
these spores can survive the
sterilization condition which can
further lead to the contamination
of the tissues. So usually whenever
there is a bacterial growth it is easy
to see through your naked eye because it
gives a characteristic appearance okay
like ooze like appearance or they'll be
having different colors this bacteria so
like green color pink color which you
can see in your fruits and vegetables
when they are getting contaminated okay
and they'll be having off uh odor that
is the smell and whenever your tissue
culture which is contaminated with fungi.
fungi.
Okay, you can see a fussy growth because
fussy growth and different appearance of
the color because always you see that
fungi have the hyper or the mycelium
growth you can see. So
So
what the scientists are doing is we have
a separate chapter on meristem culture.
They go for merist stem culture because
the apex of the chute okay the if this
is a plant and the aex of the shoot is
always free from contamination even from
the virus it is free of contamination.
So they take this part of the plant for
So what are the sources of contamination
means we already told that there is an
of microorganism because the
microorganisms also use the nutrients of
the plant and which results in the death
of the plant. The mic they exhaust the
nutrient that is they consume all the
nutrients without giving anything left
over for the plant or your expplant to
grow. And another important point is the microorganism
microorganism
change the secondary metabolites. So
what whenever I'm doing plant culture
sometimes we also use the metabolic
product secondary metabolic product of
the plant. These metabolic products can
be your either your citric acid or
certain medicinal component of the plant
amino acid the resins the gum. These are
all secondary metabolites which are
produced by the plant and it is highly economical.
economical.
Okay, it is highly useful for man. That
is why we grow these plants in the
tissue culture so that we can propagate
this plant, multiply the plant and we
can get the secondary metabolites. But
what this microorganism it will do is
whenever it is contaminating your media
it by it has all chances to change the
secondary metabolite and make it
worthless. We can't use it.
Okay. So the apart from microorganisms
we have other contamination also. has
already spoken the expplant itself is
contaminated though you do the surface
sterilization the microorganism may be
hidden somewhere within the interior of
the tissue which you are not able to eradicate
eradicate
the vessels which we use vessels means
the culture vessels which we are using
it may be a bottle or white mouth
uh flask or bottles whatever we use that
may be contaminated the media what we
are preparing we wouldn't have taken
proper precautions to prepare the media
and sterilize the media. So that can
lead to contamination. The instruments
what we are using particularly we are
going on using the uh autoclave oven pH
meters to see what is the uh pH value of
the media. So when these instruments
itself are contaminated it again adds to
the source of contaminating and finally
the environment where handling is taking
place that is your transfer room or
media preparation room. Okay. So that
entire environment itself is
contaminated. It is not sterilized and
the individual is not also is not taking
any precautions to keep himself
sterilized. It leads to a source of
contamination. That is why aseptic
condition is absolute necessary for the
tissue culture to maintain a sterile
condition so that we can proceed with
our experiment to get the perfect output.
So when we're telling aseptic techniques
the aseptic techniques can be your
chemical treatment or physical
treatment. Under chemical treatment you
can see various
disinfectants, antibiotics or here in
physical treatment we can see about the
sterilization with autoclave, hot air
oven or even radiation and filtration.
So these are some of the methods wherein
we use in the tissue culture laboratory
to see that there is no contamination or
So whenever I'm using disinfectant, how
this disinfectant is bringing about
inactivation of the bacteria? How it is
killing my bacteria? That is your
disinfectants can penetrate into the
bacteria and then it can denature the
bacterial protein or it can decrease the
enzyme activity.
they inhibit the bacterial metabolism.
So when the metabolism is occurring in
steps, it can inhibit this metabolic
pathway. So the end product is not
produced and your bacteria can
ultimately die. So they can damage the
structure of the cell membrane.
Okay, you have this these bacterias will
be having their cell membranes. it can
make hole through this membranes and
then whatever the materials are present
in the bacteria can ooze out of it
killing the bacteria. Okay, that is what
they change the membrane permeability.
So these are the ways in which
Coming to the next part
that is um here we are going to see that
how we can sterilize a plant material
that is the expplant what I am taking.
Suppose say
this is a plant okay this is a plant of
my interest for example I'm saying and
then I know that this plant is carrying
some desirable trait which is of
interest to me which is economically important
important
and beneficial for mankind. So what will
I do is suppose I take this part of the
leaf and that leaf will become your
expplant. Okay this is your expplant.
Now what I have to do I have to
sterilize it. So how we are going to
sterilize it?
Plant material should only be surface
sterilized at suitable concentration.
This becomes very important.
See to to obtain sterile plant material
is difficult because in the process of
sterilizing the living material should
not lose their biological activity. When
you're sterilizing
your aim is to discard the microorganism
and it should not affect the sterilent
whatever you're using chemical. It
should not harm your plant. If it harms
your plant, the biological activity of
your plant is lost.
So the plant plant plant organs or
tissue are therefore only surface
sterilized by treatment with
disinfectant solution at suitable
concentration for a specified period. So
you should be careful of what is the
concentration I'm using, what is the time
time
limit I'm using it. Otherwise apart from
killing the microorganism you're
destroying your biological activity of
your plant of interest. So what are the
disinfectant we can use is chemical
disinfectant. I can use 10 to 12
percentage of hydrogen peroxide for 5 to
15 minutes. Broine water 1 to 2% for two
2 to 10 minutes. 1% solution of chlorine
water with mercury and silver nitrate
can be used and absolute alcohol is also
used for only hard tissues that is seeds
your root which can withstand the
alcohol. Okay. The pure absolute alcohol
which can withstand only they can be
used. Otherwise if you going to use
delicate tissues no like this flour or
buds and all if you're going to put it
in the absolute alcohol it is going to
dehydrate the that those tissues also.
So you you should be careful what
expplant I am using, what chemical is
suitable for that plant, how much
minutes I should immerse my plant into
that particular disinfectant. And also
look at this this broine water. The
broine actually what we get is highly
concentrated and we dilute it with
water. Okay, that is known as broine
water and mercury chloride, silver
nitrate these are all what somewhat
toxic, poisonous. So when you are using
this you should take utmost care that
this does not enter into your skin and
result in poisoning of youth. That is
the important precautions we have to
take. But most of the time in tissue
culture we use this.
Okay. Like uh sodium hypocchloride
for uh 5 to 30 minutes. I can use sodium
hypocchloride or uh calcium
hypocchloride. We can use again this for
5 to 30 minutes.
Okay. Most commonly which are used. So
your sodium hypocchloride you can use it
at a concentration of 2%. Whereas your
calcium hypocchloride you can use at a
concentration of 9 to 10%.
Or 70% ethanol is used for disent
disinfecting plant tissues which are
delicate tissue. There we told about
absolute alcohol. Here it is only 70%
alcohol which can be used for delical
tissue treatment and usually it is used
for only for few seconds
and also for the for disinfecting
the hands of the users. Okay, there you
can see that you'll be using 70% ethanol
or even isopropanol is recommended for
rubbing the alcohol. Now sometimes what
happens is the microorganisms
okay the entire plant will be having lot
of contamination from the uh
microorganisms okay this is known as
systemic contamination that is the
entire plant itself is getting
contaminated during that time what you
can do is you can wash it with all the
chemicals okay after that you can rinse
them with antibiotics.
Okay, the antibiotics becomes important
if the plant is having systemic
infection. But remember after treating
your plant materials with the following disinfectants,
disinfectants,
final rinse, okay, should always be
So this point you have to remember
whether whatever disinfectants
antibiotics I'm going to use for my plant
plant
after that particular minute
you have to wash it with your distilled
water that becomes an absolute
importance for the plant not for the worker.
So we can see here
the hard expplants like root seed mature
endosperms are directly treated with disinfectant
disinfectant
Now sometimes what happened an next
plant carries a heavy load of microorganisms.
microorganisms.
So these such type of plants needs to be
washed in running tap water for 1 to two
hours prior to its treatment with the
disinfectant of the plant. So if it is
carrying a heavy load of microorganism
for 10 to 2 hours you have to wash it
with running water before you are or
prior to the use of the disinfectant.
This becomes a at most important and also
uh some of the plants like you see the
chute aex or the poland grains okay they
are free from microbial contamination
there by for such type of plant
materials it is not need not necessary
you do the surface sterilization
it is not compulsory otherwise you can
use even detergents
Certain detergents are available or
these are surfectants. Okay. Like Trton
X, Twin 80.
Okay. These are used for treating the
So what are I told you the antibiotics
what we are going to use? It can be
carbon insulin, tetracycl, streptoyin,
rafampin. All these antibiotics can be
used for the treatment of the plant if
it is necessary. But when you're using
antibiotics even that comes with some uh
demerits. Problem with antibiotics is
they tend to be selective. Okay,
selective in the sense and antibiotic
cannot kill all the bacteria.
It can kill only the specific bacteria.
Resistance to acquisition. That is
what happens is certain microorganisms
like we know like many of the
microorganisms like our body has become
resistant to the antibiotics. Though you
take you see that we we end up again in
the disease conditions because the
microorganism is becoming resistant to
those antibiotics. So that becomes a big
question. What if it becomes resistant
the microorganisms we can't use that and also
also
like uh um they may obscure the presence
of microorganisms
that is
it is very sometimes it becomes very unclear
unclear
or uncertain for the antibiotic itself
to know that whether there is a presence
of microbes or not
because these will be hidden the micro
can hidden somewhere within the plant.
So also some of the antibiotics can
inhibit the cell and tissue growth
because we are growing the cells we are
growing the tissue we are growing the
organs in in vitro condition and suppose
if I'm adding antibiotics into this this
antibiotic can interfere with my
expplant which I am growing. So these
are some of the demerits when we are
using the antibiotic. So we have to look
Coming to UV radiations as already we
have spoken about it UV radiations which
is present in it is a germicidal it is
present in the laminina hair hood or
your lab or your cabinet or even the
room you can fix it. Okay. So these are
nonionizing radiation which is having a
very high wavelength of of nanometer of
200 to 400 nanometer but the most
preferable is 254 nanometer but you have
to take the precautions that you are not
present when the UV radiation is on
because it leads to mutations.
Now next coming to the heating the
heating can be of two type okay dry heat
and the wet heat. When we are saying dry
heat, it is preferably your micro oven.
Okay. All the glass was used in the PTC
should and must be sterilized before use.
use.
And your solution, your culture vessels
are usually autoclaved. They are not
treated with dry heat. Who your
solution? Whatever the media you're
making and the culture vessels into
which you're pouring your media, they
are all autoclave and also the
temperature sensitive compounds. Okay,
like uh your growth hormones,
sorry, the plant growth hormones, okay,
they all cannot be sterilized or they
cannot be treated with dry oven,
dry heat. So what you have to do is
because they are very unstable, they
heat labile compounds. So what you can
do is you can filter sterilize such type
of compound which compounds growth hormones.
hormones.
Okay, you have you should do a filter sterilization.
So you have to take precautions. The
containers, the containers means the
culture vessels in which the expplants
are cultured are called as culture
vessels. It can be your your culture
vessels can be your test tubes. Okay? Or
or it can be any flask. Okay? So
whenever you're using these type of
culture vessels, you have to see that
or sometimes what happens is some of the
culture vessels are fitted with membrane
filters. Okay, they'll be having
membrane filters
through which exchange air can exchange.
So whenever you're starting with plant
tissue cultures, you have to see that
your glasses are clean and which glass
should go into dry heat and which should
go for a wet heat should be decided and
if they are heat label compounds then
they should be filter sterilized.
So a micro oven while we are using is
for the melting of the agar. So that you
require a special container when you're
using the micro oven. Flaming or heating
that is you're incinerating your scarpel your
your
needles or your loops all this you are
going for flaming that is 95% ethanol
in an alcohol burner this is an
instrument is useful for sterilizing the
metal instrument so all your metal
instrument goes for flaming see in the
next picture this is what alcohol will
be there
and then it can incinerate all this
Coming to
autoclaving okay in autoclave I told you
that it's generating autoclaves generate
steam. So it is steam heat under
pressure very good method even for
killing of the spores. So here you use
the condition is 121° centigrade for 15
pounds per inch.
So under this condition it can bring
about perfect sterilization it is very
fast and it is effective.
And also one problem is when you're
using solution like water or medium for
your uh PTC the time of autoclaving is
very important. You can see here if I'm
having 250 ml of solution I should
autoclave it for only 15 minutes. 500
means 30 minutes. 1,000 ml means 40
minutes. So as my solution concentration
I mean the volume not concentration as
my volume of the solution increases. You
can see that the time period also
increases for the autoclaving. Excessive
autoclaving is not advisable because it
leads to the breakdown of your organic
compounds. Your organic compounds be can
become charred. Okay. So even if there
is sucrossse or is present it becomes
caramelized. So what happens that
nutrient will not be suitable for the
growth of the microorganisms.
Coming to the filtration you can see that
that
as I told you these growth promoting
hormones like the gibbrlic acid or
indole acidic acids the proteins or
antibiotics are only filtered they
cannot go for dry heat or wet heat and
usually here what happens one simple
example I have shown you see you can put
all your uh liquid the solutions
whatever toll into the syringe and that
to that syringe a filter is fixed fixed.
Okay, it is usually a cellulus membrane.
Okay, here it will be fixed and when you
press your the syringe all the fluid
will come and get collected in the tube.
So what is happening if there is any pro
contamination in this non-sterile liquid
it will be filtered out of the by using
this membrane and you'll get a sterile
fil liquid or the media whatever is
necessary. So this is very one of the
very good method. This is a simple
membrane filter I have shown which can
accommodate very little amount of fluid.
When you're going for large amount of
solution then you can go for vacuum filtration.
So with this we can end our today's section.
section. [Music]
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