Precaution during designing the lab out – III

September 13, 2019 posted by

welcome back to the lecture series in cell
culture technology so today we are into the fifth lecture of the second week so we are
into now week two lecture five w two l five ok so in the last class so we talked about
that in this corner if we put a laminar flow hood with a sitting arrangement like this
ok or or sitting arrangement like this or maybe a sitting arrangement like this we cut
the so before lamina flow hood design lets decide one more stuff you get the animal inside
right so here you are getting the animal inside the facility now you have to have a set up
for sacrificing the animal so your sacrificing the animal means you to
kill the animal and in that case say for example we consider that ah you will be using mice
or you will be using rats or something so you remember in the last class i told you
that you needed the animal ethics committee clearance so thats where you have to get your
clearance how first of all that clearance will come with how many animals you will be
using one where you will be housing the animal two in the housing what all things will be
providing in terms of you know food the kind of food you will be supplying the water supply
the air conditioning and all those paraphernalia but what is very critical is that how you
are going to sacrifice the animal if you are sacrificing and taking other tissue what kind
of mode are you going to use are you going to kill them by carbon dioxide or you will
be doing something called cervical dislocation ok or you will be using something some other
mode so there are different modes by which you
can sacrifice the animal and these different techniques are getting revised and updated
from time to time through the years there are several improvisations which are taking
place and the veterinarians or people concerned or aware of it and they can literate you on
that that you know this is the technique which is best suited for you or for this kind of
animal or that kind of animal or this kind of or say for example you wanted to do an
anesthesia say for example you are not sacrificing the animal ah lets put an alternative ok you
want to do is an anesthesia because say for example you wanted to implant something in
an animal and you know you want to take the surrounding tissue
so in that case what kind of anesthesia you are going to follow see if you are not in
veterinarian will not be aware of it because there is no reason for you to know this additional
piece of information so this part is very critical and whatever you do should tally
with what you have promised with animal ethics people should there should not be any discrepancy
promise something and you do do the stuff different way so lets assume in this situation
then you wanted to do a co two sacrificing so that again brings us that you needed a
supply of co two which most of the labs currently across the world does you know
so and you have to have a chamber this kind of a chamber like you know where you keep
the animal and you have to have a gas supply something like this and we allow the animal
to have a peaceful death and then whatever way you wanted to dissect it out or you decapitating
the head or you wanted to remove some kind of a gut tissue or some kind of muscle or
you wanted to do a isolate the cardiac tissue you want to isolate the brain so driest will
follow but this part is very important so in this facility you should have a small corner
and prefer a corner and close to the sink the reason being because this is something
which is kind of you wanted to you know do it very cleanly you dont to mess up things
here ok so you have to have a facility in the case
of course these are all assuming that you are doing primary culture because if this
is clear to you then the secondary cultures and all will be very easy to you ok so you
have to have a facility somewhere and and these this animal sacrificing should deep
inside the lab ok even in the outer facility you should not have something of that kind
out here or out here or somewhere out here right you prefer to the have this very close
to the door ok somewhere out here or some people would they do which ah i dont know
i mean how good it is they even have something outside they just sacrifice the animal and
just bring the sacrifice animal inside instead of doing the sacrifice inside the
facility well i mean it has its advantage and disadvantage because ah the disadvantage
is that then you need another additional kind of enclosure which is much more exposed and
the dis advantage is ah that you do not have to do this stuff inside the outer facility
of your lab ok so i will leave it to your ah wisdom which one you prefer but some such
facility has to be there and my need this one this will never be written in textbook
how you do it and this is what i am telling is from my experience of setting up labs design
and layout how we really have to think beyond because when i said the first class i still
remember back in probably nineteen ninety seven or maybe earlier than that i was not
aware of it and there is no textbook which could tell me
so it is more of like you know and this was not the www dot eda of you know internet and
all those things these where like you know and so there are handful of textbook here
and there but not really telling you how really you set up the whole thing what are the points
you have to keep in mind so you can see it was very trial and error you know talking
to people and seeing the facility and figuring ah this is another right way this is like
you know so whatever i am telling you is from share from my experience there is no textbook
which is going to tell you all these things that how you should but you have to think
in advance because if you think in advance and put you sure you can think far better
than what i am putting out here for you ok and if you think it right and there are people
who are very good they can tell you that you know if we put it here this is the problem
they may not be knowing your science and they dont need to even but they can give you a
better insight how you really can design the whole thing in a way what i am trying to tell
you are those rate limiting points which most of the time we missed out and then we do some
hanky panky job you know or do it like that in a fitted fit things like that and that
really a bad design bad way of like you know putting things together so please be careful
huh whenever you set up the thing think think calm quite think over it how you really want
the facility because the your facility looks nice you can bring people and finally the
end user they will be happy you know this is a very systematic facility and one more
thing these kind of facilities takes a lot of soul to be put there
so those whose server uses this such facilities should carry that spirit of that soul that
is you know we are not going to you know mess up with because any any any negligence could
destroy the work of many other people because you are handling biological samples which
are prone to contamination they can destroy your whole facility in no time and once the
contamination takes up its very tough to contain them ok so coming back so you have to have
a animal sacrificing facility and you take the tissue out whatever concern tissue and
now you have to do everything in the hood now when you get into the hood with the tissue
you have to have a buffer system this part we will come in the medium development you
have to have a buffer system which will be helpful to ensure that the tissue remains
alive during our dissection part ok the animal is dead anyway but it has to be supplied with
necessary sometimes if the tissue is very prone to know oxidative damage you have to
ensure that you know how you can handle it what kind of additional material you main
love to put some kind of antioxidant and whatever you know we will come later into this part
while we will talk about medium developments but keep this in mind this is very important
you know so out here inside this laminar flow hood
you may prefer to have a permanent fixture or a not a fixture a permanent location for
ah dissecting microscope you will be needing this pretty frequently
if you are dissecting big animals you may not need it but if you are dissecting embryos
or features you will be needing it so see through the catalogs on through it comes about
the microscope and get a good quality dissecting microscope and here is my what of like in
a caution do not do not because this dissection process especially for the embryo is a tricky
process and do not is spoil your eyes by having a bad microscope for that purpose because
its a pain because you wont be able to locate the tissue right if you have a bad microscope
look through the good microscopes of zeiss olympus or you know leica so where these are
some of the giants in the field or nikon as a matter of fact you know to have a good dissecting
microscope and if you are really good in that then i
would recommend you something which is a very good teaching aid is that many of these dissecting
microscope comes with an attachment to put a camera which could be put on a screen and
you can record it so lets think of a situation your lab works on say hippocampal neuron hippocampus
is a very specialized part of the brain which is involved in learning and memory and most
of the people who work on hippocampal culture cannot rely on cell lines they isolate hippocampus
mostly from rat or mice embryo this is a standard practice once you will come for the primary
cell cultures in depth we will talk about it
so now in the brain isolating hippocampus is not something very easy so the structure
is something like this if you look at the structure and of the brain so for example
you take out the brain you remove the skull and everything you are having the dorsal view
or the top view of the brain it sink with the so what does that mean that means that
animal is you know sitting like this and you are seeing the animal from the top ok so now
that is called a dorsal view so this is the dorsal view of the brain and the ventral view
if we kind of twist or make the brain upside down like this like this this is the dorsal
view right and you just look it like this you just make it upside down
so if this part of my hands are dorsal view and these are the ventral views so if you
roll it down like that the ventral view is something like this of the brain ok
now hippocampus is an organ you wont be able to see from the top unless you cut it like
this and you remove this part so you have to scoop
it out that underneath this you will see a so which will be organ which will be sitting
like this and it is deep inside this is not something unless your a very thorough with
the brain anatomy and you really can it is not so easy to isolate this this is where
they hippocampus is line so coming back why i mentioned this so say
for example your lab has been working on this area so somebody is an expert who can isolate
hippocampus but this gentleman or this lady is going to leave the lab so what do you want
when they dissect why dont you record it so you have a system or this could be used as
a teaching aid for you know practical training of dissection and isolation of a specific
kind of tissue what you will be using in your culture right
so its a good practice to have a good dissecting microscope with possible attachment or camera
and ah viewer and this viewer could be in computer you can you know interface the camera
with the computer and the computer screen you can see and you can record it there is
a good dissection you can record it and that could be a tool for ah cell culture facility
that you know this is how we isolate the tissue ok so i will recommend careful while buying
a dissecting microscope and go for like i personally have use zeiss you are really good
ones really good ones i have used nikon i have even used ah leica so these are some
of the good ones olympus i have an used but you know i have heard good things about it
so but try to get a good dissecting microscope my first suggestion so you have to have a
dissecting microscope inside not here where all your dissection will take place in the
corner you prefer to have a table top sterilizer what is a table top sterilizer so for dissection
you will be needing dissecting instruments ok so be careful about the dissecting instruments
again a tool which you will be using very frequently if you are a primary cell culture
lab so try to get the best quality of it there
are global companies which are really good like fine science tools
fine science tools this is swiss company they are the maker of some of the finest tools
i have ever used till this date and if you do not find them and if you cannot afford
those ones your lab is not ready to afford those ones you can always go to the local
goldsmith and they can really curve out or the watchmakers they can really curve out
very nice instruments for you but you know you have to you know valley up with them in
order to tell them this is your requirement because most of the watchmaker in our country
they use very very fine you know fine tercets scissors
so you can get those things fabricated which is not a big deal trust me or you can get
a coarse one and then you can gold plate it and you know make it sharper and several things
you can do there a lot of zone for innovation tremendous zone for innovation what you can
work through but you have to think how you really can make these things happen ok so
get the best quality dissecting instrument and there are dissection instrument box its
something like those of you have ah seen in other lunch boxes which are made up of ah
aluminum or steel or something these are box similar to that underneath it you have a kind
of a cushion like thing which is a polymer or rubber kind of thing keep a rubber on the
side so that ah say for example you have the box like this with a cover
so you have the rubber bed like this this rubber bed ensures that the instrument which
are there so you have see this fine sharp you know forceps you have very fine ah should
say very fine you know scissors capsules and you did not want these to get damaged so what
you do you can even fabricate you can get a box and you can you know you can put very
nice rubber lining so even if the tip of the instrument kinds of get hit should not get
damage so again these are the experience which is telling you because i have seen things
getting damage destroyed just because we didnt take proper care which you could have done
all right if you have been just little bit of farsightedness so you have to dissecting
box so before you get the dissecting box what
they do you sterilize it so you have to have one one more thing you have to have an autoclave
somewhere out here now this autoclave can be outside the lab also so autoclave is something
like a pressure cooker where at high moisture and high pressure you sterilize anything so
you will be needing autoclaves and ensure that these autoclaves are according to the
requirements or number of end users you do not want to get a very huge ones which is
kind of tough there are modern days facilities where they have centralized ah um autoclaving
so just before i again get back to the dissecting thing let me talk about this autoclave stuff
so its the autoclaves could be multiple types like you know you could have ah autoclaves
like this you will see any of these labs they will have like this huge you know autoclaves
sitting on the corridors you will see most of the time on i stand like this
ok so they are bolted on to your fore side like this and here connected to the power
supply ok and here you keep your stuff and there is ah pressure gauge which ensure the
pressure and from the other water kind of you know create the vapor so in a high pressure
and in a vaporized environment you sterilize the stuff so whenever you are keeping anything
for a sterilization or autoclaving you put them in a autoclave pouches their pouches
which are available so its kind of a you must have all have seen envelopes where you keep
our send our letters or something is almost similar to envelop and you can seal them
so they come like this something like the envelopes like this you know and this is the
sealing when you seal them so there is a spot you will see after a certain point of heat
and moisture the color of that thing changes which indicates that this stuff has been autoclaved
and if you visit websites of autoclave bags autoclave bags you can see these kind of autoclave
bags they are really very interesting stuff explore it online go online and check it out
you will you will really enjoy seeing them there are whole different range of autoclave
bags which are available ok so will have this autoclave bag so whatever you want to sterilize
you keep them inside the and they come in different size and shape mostly different
size and shapes ok so you can have bunch of them already purchased
for your facility and you keep your stuff in them and you bring it out till you reach
on the laminar flow hood or just close to the laminar flow hood you do not open it ok
so you get these instruments autoclaved and bring it inside your facility like so you
are out here so now as i told you that either you can keep the autoclave outside or you
can keep a table top autoclave somewhere out here especially for this kind of facility
which i personally prefer a table top autoclave which is in your control it is not outside
it is not bothering anybody you have a small table top autoclave and these are all available
they are of kind of you know like one feet size or something like this and they are very
cool stuff to you know work with this doesnt create much problem
so close to the sink because you have to fill water and everything and they need a power
supply and thats all you need it so you get this instrument inside and while you are dissecting
or automatically you may feel like you know if i could restore lies because from one animal
to second animal from one embryo to next embryo so for that you need dry sterilization which
is a table top sterilizer so these have these are sterilizer something like this they are
small like water feet stuff like this and there are lot of ah glass bead kind of a stuff
in them and you keep the instruments in it and you have a panel like this and it is connected
to the power supply ok and this dry sterilization takes around ninety seconds to a minute
so while you are working you can re sterilize but there is a difference this is where you
have a wet sterilization wet sterilization which is far more long term stuff and here
inside the hood you have dry sterilization where you are only utilizing the heat you
are not utilizing the moisture or the pressure into the game right so you have the dry sterilizer
sitting out there see you keep your dry sterilizer there so while you are dissecting you can
always you know re sterilize it in ninety seconds and it still you can use
so these are some of and of course you wanted to have a bunch of pipettes which most of
these labs used has dedicated ones for this kind of dissecting hoods generally i personally
used to prefer on those glass walls on both sides to have the pipettes can be kept something
somewhere like this you know you can have system where you are not exposing them out
and you have a sterilized pipette tips which are kept inside something like this if the
pipette tip sitting there on the bench which time to time are sterilized and every time
you use somebody comes with a fresh pipette box pipette tip box and kept it there and
remove it and again replace it some new pipette tip box
so these are some of the basic basic things what you prefer to have inside the dissecting
microscope or dissecting laminar hood so lets summarize what all you need to have you need
to have a microscope first a dissecting microscope of course if possible connected to the camera
and a viewer with a computer or ah some ccd device where you can monitor second you have
to prefer to have a dry sterilizer third a dedicated pipette and pipette tips sitting
out there so now we added one more component into it the laminar flow hood in outer facility
ok so and where you wanted to put the autoclave
so my preference will be have an autoclave inside a small table top you can have a big
autoclave outside maybe on the side of the corridor or somewhere but a small ones out
here may not be a bad idea ok and you have the water supply we talked about it so from
here in our next class we will further build up the story how this whole facility will
eventually look because we have just started on the outer layer and there are few other
things what i am going to highlight here before i move into the inner chamber or the inner
culture room where other things will be taking care ok so thanks for your patient listening
once again we will continue in the next week for the development on these ah layout design
and instrumentation thanks

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