hello fish - welcome to another Friday

night

we've made it it's been a long week for

me I don't know about you my name is

Jason and Yoshi red fish blue fish thank

you so much for stopping by and visiting

me this uh dampen ain't gonna stop

Friday evening I don't know if the

weather has been crazy for you guys

where you're at drop me some chat there

let me know what's going on but it has

been insane in the membrane here it is

driving everybody insane and it just

will not give up so but we are still

here we're still ready to rock you guys

are with us I see a few people in in the

room Philemon

Lucas Brad summer and lrb aquatics you

was doing I was finishing it up he might

run over a little bit who knows there

could be hopefully there'll be a lot of

people filtering in have a really

interesting type stream for you you

might notice from the video there's kind

of a kind of a border around my head

there is kind of a whoa let me look at

myself a great black thing that was

finish you guys because we beat up

hopefully there'll be a lot of dancin

phone rings Oh
let's have an interest rate I am

flustered it you might notice from idea

week at work there's kind of a kind of a

border buddy what's going on Caitlin

thanks for stopping by we got reels

tang's reels tanks in the house

Caleb says mild in my hair - that

actually surprised me man your way on

Easton as it must be a little temporary

yeah it's crazy on here it just keeps

raining let's not you can't call it rain

[Music]

starts to give up start to you might get

like in summers with an hour or two

bracelet it just keeps me

okay can you guys please let me know and

if the echo is way better I just muted

this video I'm watching it back watching

the chat for you guys let me know yay or

nay Caleb says something is wrong with

the audio can you guys hear me

can you hear me at this point I muted a

video sorry another device that I had in

the background and okay reels tank says

better nice

so no more echo good appreciate that

guys we're gonna do our AV check I don't

want to be sitting here for an hour

talking to you guys sounded horrible

appreciate that um we have an

interesting show that you know you guys

know that I'm a big plant guy I'm a big

aquascape guy big aquarist

fish guy shrimp guy you name it I love

it all but I recently gave you guys a

little look into a kind of a mad

scientist's room that I put together for

myself it's basically a plant tissue

culture lab is what it is it's a clean

room there's a lot of equipment in there

we're gonna head in there a little while

later and kind of familiarize ourselves

with concepts and equipment certain

terminology things to look out for but

in order for any of that to make sense

we do have to do kind of like a little

bit preliminary we need to I'm not gonna

go real deep but we do need to talk

about plant to culture concepts and a

core at a core level you know kind of at

a 30,000 foot level we don't want to go

real deep and get crazy because I could

probably sit up here and talk forever

about plant micropropagation because i

find the science fascinating and I find

plants even more fascinating than the

science so double trouble

we've got someone coming into the room

you got looks like recons aquariums

thank you for stopping by I appreciate

that

still looking good on audio I think nice

okay cool

so let me switch over to OBS studio and

do my magic you're gonna see me yes mom

but I will still be speaking

hopefully hey let's have a stroke

sarong windows sorry guys

all right there we go finally got it so

here we go some some nifty graphics let

me switch back over and make sure you

guys can see it and switching to youtube

there's a bit of a lag as you guys know

YouTube's always like just I find

make sure that the scales correctly I

think we're going to be good

check and chat while this okay cool yeah

all right guys you are good to go this

is introduction of plant tissue culture

this is core concepts we're going to do

steak you know keep it fairly basic but

there's some things when you know we're

gonna do this tonight not tonight

because we don't have time to dig into

everything tonight to to to give an idea

of what it takes to start to finish with

you know explant material ready to cut

and then all the way to finish

you're not talking maybe on an easy

plant just doing a few cultures maybe

two and a half hours

because of all the cleaning in the

sterilization and stuff so anyway let's

get started advance this presentation

here oops hey you alright I am NOT going

to torture you by reading through this

entire thing it is more for you to look

at well I talked about it and it's also

kind of a point of reference for me to

glance down at the screen if I get lost

as I as I regularly do yeah here we go

so plant tissue culture you guys have

seen plant tissue cultures you've

probably purchased them from a TA or

tropica or someone like that you know

that they are little itty bitty tiny

plants

but what were they before that well they

were ex plants and before they were ex

plants they were donor plant let me just

make sure I'm coming through okay okay

great yeah they are so the little tiny

is my boots and stems and leaves and

everything those are called plantlets

there are really really tiny small

plants they have all their organs they

have roots they have the vascular system

all that stuff what were they before

that well they were stands

what does explant it put simply it's a

piece of a plant that's it could be a

maybe a single cell it could be just a

few cells it could be an entire piece of

a plan

entire branch or leaf or whatever or it

can be like an entire organ from a plant

of certain plants you tissue culture by

harvesting root material from them so

you might hang off their entire root

that's ex plant so I think of ex plant

as ex-wife

soif dollar used to be plant no longer

why isn't it planned well we've got a

complete planets missing organs it's so

that's what we harvest of donor plants

or mother plants and we use them in

plant tissue culture so you'll see in

the text in front of you there's some

there's there's a word I want you to pay

attention to I have to talk about it

because if you don't understand that you

won't get any of these ok all plant

cells this is really important our cells

are not this way all plant cells have

something called totipotency

as you can see in the text in front of

you that merely is the ability of any

cell in a plant any cell transformed

into any other cell any other kind of so

so a cell from the tour it could turn

into the cell of a stamen you absolutely

can do that you could take leaf tissue

cells you can by way of a callus culture

can turn it into callus and it can turn

into the entire you know top part of a

plant that's a very common way to tissue

culture things so it's really important

that you understand this concept

totipotency any cell in a plant given

the right conditions can turn into any

other kind of cell from the same plant

if plants didn't have that ability

tissue culture wouldn't work

micropropagation wouldn't work we'd be

doomed so kind of scan over there yeah

tody Popes have you talked about that we

talked about what x plants are i explan

sir pieces of plant so it could be small

pieces could be little pieces so

basically X plants are tissue culture

using a handful of things you have to

have almost also known as toe hormones

also known as plant growth regulators

they have a lot of different tags that

people pee

use different names and they're pretty

much all the same thing we're gonna get

into that you need vitamins

macronutrients carbon source amino acids

other various organic nutrients you need

that and you all need it in aseptic

conditions meaning sterile sterile

sterile you're working with hormones all

kinds of stuff the media that we're

preparing that we explain us on to kasi

there must remain sterile otherwise

you're going to get contamination like

crazy so that's kind of a really over

tissue culture it's essentially a way of

turning a little tiny piece of a plant

given the right time and technique into

thousands and hundreds of thousands of

plants if you if you desire to do so and

indeed people are doing never know

that's how a lot of our food crops are

raised so let's move on

actually before I move on let me go to

Chet and make sure I am you know all of

that audio cutting out some red fish

blue fish okay thanks candy sorry about

that

let me see if I can speak a little bit

louder does it seem to be kind of like

I'm seeing too quiet situation please

let me know guys ASAP so I can shout at

the phone
hey um how about audio now can you guys

hear hopefully I will sticking it chat

for a couple more seconds if you let me

know

Tyler's over later in fact let's get

let's continue on

let me sorry let me make this window a

little bit my video window a little bit

smaller so you can see the text yeah we

go that's better

how about that okay um hopefully you

guys can make the audio out okay I seem

to be having some audio problems it was

just a bit glitchy over here okay cool

cool cutting in and out yikes that's a

burn sorry about that guys hopefully we

can hang in there so plant to us I guess

maybe it goes down there for you to

represent the steps least temple you

select your ex plant from a donor plant

you dissect it you wash it you rinse it

you perform surface sterilization what

does that mean well we washed it what

does washing plants mean how do you wash

plants you know with a washing machine

no very gently you wash it with a

specialized turned and sable brushes

brushes and a little bit of water a

little elbow grease into it you you

actually wash the surface of the plant

you rinse it very very well with still

then you perform a surface sterilization

that's basically immersing or placing

the X plans the pieces of plants into

some kind of a sterilizing compound it's

a very very common that I'll eat you use

bleach found it's not a good idea it's

very very harsh on aquatic plants I

don't like bleach I use some other stuff

that I'll show you a little while

next under aseptic conditions now this

is where it all changes

wonderful irritation and of that tissue

culture you got to have sterile

environment right hey I see more people

coming in before mad guy thanks so much

for stopping in mr. Zen so many new

people see ya you're already you've

already performed your surface

sterilization now from here on out you

got to be a sceptic you got to be

sterile you perform further trimming of

the explant if that's necessary easily I

don't and then you place the explant on

to media for initiation in a culture

that's you start that stage

yes for 20 sometimes 30 days it takes

stage one to get going that's the

initiation stage that you move on to

stage two you've initiated your culture

you've got a lot of shoots coming out of

your your explant material seems to be

doing well you said do the same thing

again

Yuri culture it Enrico true Andrey

culture it and multiply that out and

sterile petri dishes you start off with

four dishes or eight or ten however many

of you you felt like doing and you

continue multiplying those and it's in

the same fashion and a vegetative growth

phase if you will and that stage two

stage three is basically where you've

multiplied all of your cultures you

still don't have plants guys they're not

plans they're not complete plans

they're not even plantlets yet there are

no roots they're missing organs so they

can't survive yet it's time to move into

stage three which is your rudy phase now

some plants especially aquatic plants do

not have a stage three they don't have

it they don't need it you don't really

have to show them any special treatment

they will grow roots as you well know

you can chop plants all over the place

and put them all over your aquarium just

explode roots everywhere right so

sometimes with some plants you don't

have to do that but I definitely do so

the routing stage is stage C and that's

where you move you're not quite yet

plants on to a new kind of media that

has a different combination of hormones

in it hormones that induce rooting as

opposed to vegetative growth and chute

multiplication I'm gonna simplify it

after that uh about thirty days your you

do now have plant list you have

completely plants they have grown roots

the plant growth regulators the hormones

have done their job and they're ready to

be moved into a greenhouse or sold like

that if you're a DA or tropica or you

know a handful of other people basically

there is no stage for for some for some

aquarium plan Stage four is you buying

them and a little tissue culture cup

just like this right this is basically

finished up with stage 3 and this is

this this is the stage 3 container

that's what that's what happened in here

all these roots you see crazy roots all

over this is literally the container

that was used to move from stage 2 to

stage 3 they just covered it with nifty

cellophane and slapped a label on it and

charge you a lot for it so that's kind

of the tissue culture two-step process

again at a 30 or 40 thousand you know

foot altitude level without going too

in-depth right now let me switch back

over to chat see what we got going on

hopefully the yeah is gonna stabilize

and freaking out anymore oh we got

peplum Creek aquatics in here peplum

Creek aquatics thank you for stopping by

we've got a kwatak ness in here

forty-four mad guy fishing more or candy

as always punchy paints thank you punchy

for stopping by really appreciate you

guys being here Roxana hey Roxana a new

kind of a new subscriber really

appreciate you coming by hopefully you

find this this informative so let me

switch back over and we'll advance and

keep on moving forward this sounds kind

of gnarly right now trust me I'm going

to really simplify it next slide

you don't need to soak this in this is a

list of well-known offices just to kind

of show you how many this is far from

all the ovens that we have to work with

i'm autism's

are used for well to that in the next

slide you can see that we have a lot to

work with we have a molecular way that's

very important we have you moles for one

milligram per liter what it so you know

what what it what is all then that's

what solvent means diluent what you have

to use to dilute it should you need to

dilute it is it powder how do you store

it sterilization methods we're gonna

spend some time with that that's super

super important working if we don't care

about that

that column it's it's rarely correct but

just check it in chat okay here we go

sorry guys okay so I want to talk to you

about sterilization you see that column

there second from the right

sterilization we've got some symbols we

got CIA F and F you see the little

legend at the bottom see a is Co

autoclave herbal with other media

components what does that even mean that

just means that that substance can get

really hot like really hot 240 250 260

degrees for a good half hour and it

doesn't degrade it doesn't break down it

doesn't it doesn't mix with other

hormones or substances it's stable and

it won't break down and heat but if you

know about actually just above boiling

temperature they can handle that for the

30 45 minutes so it's kind of label a

problem F we see F if you see F that's a

bummer

that's filter sterilized that means that

that substance absolute cannot tolerate

heat at all if you slap it in an

autoclave and media was on hormones and

era sugar charcoal whatever you're using

myo-inositol maybe and you apply heat so

it's toast it's done its history it

cannot tolerate heat but those have to

be cold filter sterilized as there as

there as we say to keep it simple it's a

liquid hormone that you load up into a

syringe and you hook up to this little

device with a term in it and you force

it through that filter the filter is so

fine and filters even bacterium and

bacterial spores out simple but those

filters are expensive and it's a real

bummer to work with it that way so we

always want to be able to autoclave what

we work with because it simplifies now

CAF kado sorry kono claimable with other

media post however some loss of activity

may occur

that's where we usually were CAF it's

kind of playable but there may be some

loss of activity in the strength of the

hormone of the dogs in the cytokinin

whatever you're working with

so you just account for that and you add

a little bit more it's that simple if

you can get away with it going ahead and

cold filter cold filter it out that's

that's better to do and then let's kind

of jump forward I want to spend too much

time on this there's a lot of them and

are well-known cytokinin again a lot of

them a lot of weird ones actually let me

go back and show you guys something do

you see down the well-known list of

auxins I think it's one two three third

from the bottom

Pickler on a little a little history

about that that is actually a component

of Agent Orange yeah we don't use that

we don't like that in fact that stuff's

banned in Europe so there are some

really great hormones some phyto

hormones some eggs and some cytokinin

there's there a couple really bad ones

that we stay away from here's our list

of Saito cannons as you can see there's

even more of cytokinin and some of these

are definitely artificial man-made they

don't occur in nature and then we have

miscellaneous plant growth regulators

Jay's monic acid is is pretty common

Garelick acid even more common we use

that a lot and you can see give a row

like acid is ko autoclave able with some

loss of some loss of strength but I

autoclave it all the time it's fine

and here we go so let me take Manisha

I'm gonna leave the text on here and I'm

gonna go back to chat and make sure that

everybody is able to kind of follow

along with what I'm talking about so far

I really hope so I don't see anything

people asking questions already in chat

which is a good sign

I'm trying to keep it you know pretty

high level at this point we got some

more people coming in and we've got

father fish welcome father fished

thank you so much for coming by it's a

real pleasure to have you Keeler's

aquatics in the house Taylor's aquatics

in the house rocks and as a house nice

more and more people filter and I said

really appreciate that guys so I will

keep jumping back and forth between text

and chat we do need to talk though about

Austin's vs. cytokines hey this is very

important stuff you don't have to know

all the August ins you have to know all

the cytokinin you just need to know what

they are and why they're different

because they're very different if you

don't understand the difference between

you're gonna have a really hard time

with micropropagation

so first you see some bold print and

then in red text a core concept anything

bold on this page pay very very close

attention to it's important okay Ogden's

are growth hormones there are phyto

hormones they're naturally produced by

plants as it says nor when the chute

tips and the root tips

generally speaking auxins increased

cellular division they do a lot of

different things however and this is the

bold part I'm gonna read this and please

pay attention the most desirable trait

in Ogden's in plant tissue culture is

their ability to initiate the formation

Ogden's are routinely used in plant

tissue culture to initiate root

formation on both dissected stems and on

callus Ogden's also have an elongating

effect on developing shoots in plant

tissue culture that's super important we

use it for rooting and they also

elongate developing shoots and that's

what we use Ogden's for primarily can be

used for other things but that's really

where we where we use them in plant

to culture and micropropagation Smoove

on the cytokinin okay cytokinin czar

very specific they're a special class of

phyto hormones little trivia I threw

some in there the term site under the

name derives from the term settle

Kinesis which is the process of cells

dividing into many many cells because

that's exactly what Saito canons do of

cellular division but they do more let's

jump down to the bold part the most

desirable trait of cytokinin is that

they act as attenuators were blockers

against the shoot along eating effects

of oxen's

while promoting an increase in shoot

development and blood proliferation you

might be going hold on one something

elongates something and something blocks

something and what these these two

things seem mutually exclusive they're

not actually they're contradictory but

they're they don't cancel each other out

I try to set up in the core concept and

the next slide is going to totally make

sense to you guys

auxins versus cytokinin core concept

here you got to paint into this Ogden's

typically used for routing and shoot

elongation and sightedness

typically use development and blood

proliferation directly contradict each

other in plant tissue culture yet act in

concert to produce a complete yet from

ex plants

okay they were together although they do

contradict each other that's why we have

different phases stage 1 stage 2 stage 3

stage 4 yeah see we're playing with the

ratios of augs and vs. cytokinin - and

do in certain behavior and in this

tissue so let me jump forward one one

more widen okay here's the one that kind

of read us few guys out definitely

I saw something similar to this before

and I thought yeah I'm gonna have to put

like that together I don't that I did so

we've got this this chart in front of us

and you can see that there are what one

two three four five basic you know sort

of horizontal levels that go back and

forth on the Left we have Ogden's on the

right we have cytokinin

okay you can see that the top of Auden's

is kind of a you know kind of a triangle

at the top is where it's the most

powerful and as it tapers down at the

bottom that's you know it's getting

weaker in effect cytokinin is exactly

the opposite mase we try covert chancery

so as you go on this this this graph

here and you select okay what I want to

do I want to start I want to start a

tissue culture I don't want it pelipper

a couch I want to do it the other way

around you own I want to create shoots

first I wanna I want to create Reuters

you do I want to create Calais just

tells you where you basically where you

start so you see if we more or less

balance

Ogden's plant growth hormones or phyto

hormones known as oxen's

against cytokinin x' right you see that

where it says callus induction that's

exactly what's gonna happen we're gonna

induce callus we'll talk about what

calluses in a second if we want routing

on shoots very high on the augs and and

virtually no cytokinin routing on callus

adventitious buds shoot proliferation

you see how as we move up and

depending on what do we have to play

with our ratios of ogz and versus

cytokinin I hope that kind of helps you

guys understand how these plant growth

regulators and plant growth hormones of

work okay we're going to move on ahead

and jump into basic you come home what I

mean what's media it's this stuff it's

that that jelly stuff that we grow stuff

on sometimes it's liquid sometimes it's

jelly yeah liquid cultures are very very

popular tropica uses liquid culture

cultures pretty much exclusively there

are a lot easier to do with some plants

anyway let's jump into the basic media

components I don't want to spend a lot

of time on this we're already and we

already have a fan out of this it's

insane

macro-nutrients you got hydrogen you got

oxygen you got nitrogen phosphorus

potassium calcium magnesium and sulfur

okay macronutrients macro big - iron man

manganese I always do manganese zinc

boron copper and molybdenum micro micro

macro my what makes it matter what makes

it micro why don't why are some mackerel

and some micro I liked somebody to drop

it in chat I'm not gonna give you the

answer right now

why do we call these micronutrients and

those macronutrients and the others

micronutrients it's the difference any

of us you're probably familiar with some

of that Fido if I am in you definitely

are niacin vitamin b6 and myo-inositol

those are the same vitamin vitamin czar

we use those vitamins they're totally

viable for mammals not just plans we got

our amino acids casein Hydra hydrolysate

l-glutamine él espera gain and adenine

adenine is very very commonly used

all the time carbon you need a carbon

source right plants love carbon why do

you think they're breathing co2 all the

time in they're getting carbon yeah you

need a carbon source

these plants when you chop them up and

you put in these two cool cups they

don't have a vascular system anymore

they're not a plant anymore so they

can't breathe can't respire so if they

can't respire how the carbon sugar

that's how they get carbon we feed them

sugar normally sucrose almost sucrose

rarely fructose almost never die straße

I rarely see that and charcoal is

actually an excellent source apartment

and it's used for other reasons I'll

explain that in a while

phyto hormones you see my symbol

infinity not gonna go into that again

various beneficial organics double

infinity tons of them can't cover all

solidifying agents agar gel right etc

and you see sometimes that's optional if

you're doing a plotless you may want to

do liquid medium you may want to do

liquid tissue cultures and never never

really never never solidified and just

be happy with that I see someone is

answering some questions I just had it a

second ago who was it

oh the things ginger said particle size

and absorption close

it's actually close macronutrient versus

micronutrient actually refers to the

amount we use so if we use over a

certain amount

it's a macronutrient they use tons of

nitrogen tons of oxygen tons of

potassium all that they use very very

little of it under a certain amount as

classified as a micronutrient that's the

real difference the same applies to your

liquid fertilizers guys you're dumping

in your aquarium you're drinking your

tropical your whatever you're using

maybe your fiction you're not doing that

at all because you were doing the deeps

okay we all are digging the deep

substrate we'll get on that in another

video but my macronutrients vs.

micronutrients same thing applies not

just in tissue
culture is it's everywhere that pretty

much covers basic media components

that's pretty much it I thought I saw a

question in here let me jump over and

answer a question from Roxanna questions

so if you get a plant culture and it

doesn't produce roots that means it was

lacking the Ogden's not necessarily not

necessarily that might mean that they

never they didn't even bother putting

putting it into a rooting stage it might

have been a young tissue culture - they

may have put it into a rooting stage as

like whatever that's basically almost

all these tissue cultures by the way

because I don't know if you know this

India they're all made in India pretty

much Indians are some of the most gifted

plant scientists when it comes to

micropropagation ways smarter than me

way smarter right maybe one day I'll be

that smart smart as these plant guys are

but I'm just a I'm kind of a crawling in

my in my owner burry at this point um

sorry going over chat real quick you

guys chat amongst yourselves

yeah Roxanna yeah yeah yeah well Roxana

says it is hair grafts will it get roots

oh you betcha

oh man paragraphs will root like crazy

it's got meristematic regions all over

it it'll grow roots don't you already

one bit that's actually very very common

to get root continued roots to get a

tissue cultured like really small plans

like mosses and little grasses and small

plants like that it's very very common

to get them and pull them out Billy hey

where's the roots sometimes they don't

have any they will absolutely lose so

Chattanooga ed says I don't know

anything about microbiology well that's

okay dad that's alright that's why we're

all here I just I just you know I I

stayed at the right hotel last night you

know I'm not gonna say the name because

they're not paying me but I stayed at

that hotel last night I'm just I'm just

playing one of these guys on TV or on

YouTube anyway let me hop back over to

the presentation and we might be at the

end ah look at that we're at the end

well we see that we have arrived at the

intersection of lost confused unsure

unclear perplexed disoriented bewildered

and knowing me probably could use a

shower no I'm just kidding I just took

one so let me switch back over to chat

and we were just kind of answering

questions let me scroll up there's a lot

of people in here thank you so much for

coming by by the way I'm gonna scroll

through these really really quick and

see if I can see if there are any

questions and I will just keep pushing

forward

hopefully my audio is stabilized guys

I'm really sorry about that I've really

been struggling with audio it's

something that I need to address I'm

actually using a I've got some software

on my phone that turns it into like oh

damn sometimes it's great sometimes not

so much just going through I think

there's a lot of comments okay so I

think I'm I think I'm good I think I'm

good

um so are there any specific comments

for me regarding plant tissue culture

what we what we've covered so far if

there are now is the time to bring it up

before I hop into the lab and I start

showing you around a little bit

familiarizing you guys with the with the

equipment in there with some of the

concepts back over to Chad's doping in

and out for me gosh that's terrible

really sorry about the audio guys is

everybody am i cutting out on audio for

everybody or just candy please let me

know in chat while I watch chat

[Music]

how's the audio guys looking good

hopefully waiting for chat to catch up

with the video that's always annoying

father fish is ready for the laughs well

I suppose 20s I don't some gosh we're

just gonna have to push ahead guys

hopefully hopefully it's not too bad

bear with me now I won't be able to I

will not be able to see chat while I'm

in the lab so bear with me guys let me

pick the tripod up start moving flip the

camera around sorry bear with me guys

how do you flip this camera

okay

this is the lab holy video isn't to echo

in here this is essentially the

cleanroom let me put this tripod legs

down guys bear with me a little shaky

shaky I will refine my camera skills as

time goes on okay so this is a plant

tissue culture lab this is essentially

the sterile the sterile work area this

is where work occurs sterile work I

should say let me set this tripod down

here and this is a sterile flow hood

this is I believe it's four feet by two

feet the concept of the sterile flow

hood is that air intake happens in the

back you can't see it but there is space

on this side and space on the far side

where unclean you know septic air is

drawn in with two really powerful

turbines and then they're forced through

this high-performance filter this is a

HEPA filter it filters out pretty much

everything bacteria bacterial spores

fungal spores viruses nothing gets

through it at all so the company the

idea is is that you've got your

equipment here everything is sterile you

switch on your flow the first thing that

happens is there's tons of bacteria and

viruses and stuff that get blown off of

it right so it's you have to sterilize

once you turn your flow on but once you

turn your flow hood on you sterilize air

everything down with alcohol you make

sure everything's nice and clean and

with your flow hood on running the whole

time once you start this process you

cannot turn that flow hood off because

once you do unclean air rush

in right the fluids constantly putting

out sterile air constantly it never

stops until you turn it off

so unclean air never has a chance to

close in on this area where you're

working so effectively whatever is here

stays sterile whatever is back here

behind it is not sterile

so it's really important you understand

how these things work

yeah I can turn it on and show you guys

it's a little bit loud

[Music]

and I'll check chat I'll be right back

okay guys I'm back appreciate that I

went back and checked the chat and I saw

somebody asking can you please make it

the window bigger and I just did I'm

sorry I forgot about that before I

walked away guys anyway yeah this is a

laminar flow hood actually this is not a

laminar I purchased this from a company

in New Jersey

Ellen air technologies they're a

fantastic company company to work with

if you ever want to purchase the

purchase of flow hood they're fantastic

they were really great to work with

let me collapse this again and sort of

walk around

sorry for the shakiness

almost there one more leg

okay so that's a laminar flow hood air

is drawn in back here

in fact this is a pre-filter that I'm

pointing at here can't show it to you

this is not for drinking by the way guys

this is fuel for an alcohol lamp

that's not my secret alcohol stash wink

so yeah this is a laminar flow hood I

can feel the air hitting me now this is

very very important this is how

basically I ensure that from right about

here right to about here and out to

about here all the way across that whole

area all the way up to here right all

the way up to here that whole imaginary

rectangular area about to about to here

as long as that stays on there will

never be any bacteria viruses creepy

crawlies in there and we will not get

contamination at least from the air at

least from the air let me turn this off

because it's loud and it's kind of cold

[Music]

as you can tell that thing does make

some noise okay so when we're preparing

medium right that's that that's the

stuff that we're gonna tissue culture

plants on often we use these containers

these are a special tissue culture

autoclave herbal container you can

pressure cook them you can boil water in

them you can do all kinds of stuff with

them they don't melt they're very very

durable and as you can tell very very

rigid so anyway when we're getting ready

to start creating our media remember we

went over some things that they need

what were those things macronutrients

micronutrients vitamins right what else

you guys remember carbon we need carbon

Hagen cytokinin various organic

mumbo-jumbo and fairy whispers actually

not very whispers so anyway when we're

getting the meteor oh and agar and Jill

write whatever you want to do to

solidify it so when we're doing that

this is what we do that on this is

called a magnetic stirring hot plate

okay basically it can stir it's got a

magnetic stirrer built into it it uses

magnetism and this is the heating

element and see heat stirring I'll show

you how these things work you absolutely

have to have them if you're doing plant

tissue culture oops sorry

now how does this thing stir water well

let's turn it on no look nothing's

happening why well it's a magnetic

stirrer remember I said that and you

need these are called magnetic stirrer

rods let me get one of them out

pardon my shaky camera skills and get

one of the medium ones a medium one why

not all right

looks just like a little pill and it's

got iron in it basically just drop it

all right you see it snugged up right to

the middle right to the middle that's

because there's a magnet there it's

stuck right to it so we just stir it up

as you can see you can get them stirring

pretty fast if you turn it up you can

really get quite a quite a ripple going

with these things and then of course

your heat is separated this is just tap

water by the way just for a

demonstration so this is what holds your

your thermometer I don't really use lab

thermometers I like the digital ones

these things these are great 72 degrees

and work fantastic so a lot of your in

fact virtually all of your media

preparation is occurring on this device

you can boil stuff and cooperstown melt

it and stir it you never have to get

your hands on it it's particularly

helpful if you find yourself in the

unlucky position of having to create

media and work with it after it's come

out of an autoclave
right you have to add hormones to it

that were susceptible to heat right so

it's it's it's particularly helpful to

have the ability to stir and not have to

touch it with your hands the other

device that we use for creating media is

this this is a digital scientific

milligram stale scale that's very

accurate comes with these these little

essentially their breeze boxes

imparts these scales are so incredibly

sensitive that just the air conditioning

on our house on the other side of the

room can make them go off they can

actually affect them that much so these

two pieces of equipment we use

exclusively to create media right the

stuff that we're growing tissue cultured

plants on the magnetic string hot plate

and our milligram scale you've already

covered this full hood this is what

basically provides us with a clean area

here no no contamination okay obviously

tons of glassware come with this sort of

setup but a huge part of this lab you

can't even see right now it's here these

are autoclaves this is this actually

what the um this is what we sterilized

in this is what we this is what we use

to sterilize everything from scalpels to

forceps to dishes jars whatever and this

is also what we cook our media in our

plant tissue culture media got two of

them here they hold a tongue typically

with these guys depending on how much

media you're cooking up a general rule

of thumb is you know the least that the

smallest amount of tissue culture media

that you want to prepare is usually one

liter with that said one liter you

usually want to autoclave it for 30

minutes at 15 psi that's the general

rule of thumb if you under autoclave

well obviously you risk getting

contamination okay if you over autoclave

this is really important if you over

autoclave you will end up with the

really not very set your gel will not

set very well it will set and it will

always be kind of like jiggly and runny

in it it won't set very well it's

because it's overcooked overcooking

anger especially is a major problem so

anyway that's kind of touching on

a little bit see if you have pressure

gauges and all that good stuff so a

major challenge I want to touch on this

lady actually let me set this down so I

can show you what I'm talking about a

major challenge working in front of

these Flo hoods is that you have just

over two feet of space and you know it's

four foot long however and you are

finished

cooking your media or sterilizing your

tools or whatever it is do you have that

much room once you open this up let's

take this counterweight off and get this

open and you'll see what I mean pardon

pardon okay now you see what I mean

those things are big and that has a

really big flow hood - that's not a

small one though these things are

massive and you see they hold a lot in

fact I think I measured it before I

think these things easily hold yeah like

three levels of these of these things so

you can really pack these guys quite

tight now the challenge is this imagine

with me if you will you've got this

thing finished you've got all of your

stuff cooked in here it's all nice and

sealed in these little guys you got tons

of them in here right and you've also

got some large containers of water right

because in order to wash plants and have

them sterile you need sterile water so

you got a bunch of these in here right

it's very challenging at times while

this is ripping hot you have gloves on

in fact these gloves on let me show you

these hard core basically melt proof

fireproof gloves that are even chemical

safe when you're working with these

and you've only got that much space and

you've got to keep it in front of this

right because clean air is coming out if

you lift it that high you're done it's

contaminated all that work wasted so

it's very challenging to keep stuff in

the clean zone this is fairly easy but

sometimes we work with things that are a

little bit too big or almost too big and

when that happens well you usually hear

me curse a lot I think when that happens

you have to get a little creative so

instead of pulling things straight out

you have to start tilting and working

things out this way so it can be very

challenging working with these pressure

cookers are rather these autoclaves

particularly cuz you're under so much

pressure really eyes to keep things

sterile because all it takes is one

bacteria all it takes is one whiff of

air all it takes is one mistake really

just that's that's it one mistake and

you've got contamination and you're done

throw it all the way start over there's

no coming back for me only move this

down put this back up okay what else can

we talk about there's a lot to cover but

I don't want to do too much on you guys

and overwhelm you let me let me walk

over to to chat thanks yeah

hey there we go that's better

okay here we are

Wow 825 that's almost been an hour I

does not feel like I've been speaking

for almost an hour guys I really hope it

has not been too boring and that audio

hasn't been too dreadful I'm really

sorry that audio has been a problem I

hope you guys can forgive me

I just really struggle with audio for

some reason no matter kind of what

solution I choose I just can't get that

right so I'm gonna have to keep doing

some some research let me go back to

chat and see if anyone had any comments

rude comments not you guys you gonna be

okay this is where I was Oh father fish

poor poor pee pee pants alright nice I

like it

Oh marilla's yeah father fish needs a

timeout no I'm just kidding he doesn't

need a timeout
okay going through Chad a good good job

candy Roxanna says I can see again I was

breaking out the magnifying glasses oh I

do have to show you guys one more thing

in the lab I kind of skipped up

man you guys are crazy looking at me

dammit reading this this Chet you guys

got all kinds of crazy stuff going on

Chattanooga says I wish you was here he

is super smart well let's let's get him

in here man tell him dude you miss it

out get in

luckily this you know this is live right

now but it'll be available and we're

almost already at our hour before we run

I'm gonna run just a little bit over my

hour guys this is we haven't even really

scratched the surface of like slightly

advanced tissue culture there there's so

much to cover a lot of its going to come

together when I start working with it

the the main thing that was so important

that I wanted to share with you guys is

the role of auxins and cytokinins in

plant tissue culture you use them both

they each have their own role they

contradict each other but they don't

cancel each other they're used in

different ratios to go from rooting to

you know all kinds of different things

so we have a couple minutes left let's

go back in the lab I wanted to show you

and really show you plant tissue culture

nerdery stuff oh here it is I made a

boo-boo when I show you guys did Lulu

actually had to spend some money

last a minute okay here's the plan I'll

be honest with you guys my plan was that

there we go

my plan was is that we were going to be

doing some tissue cultures tonight we're

actually gonna be jumping right in and

we're gonna do like 30 minutes overview

like really flight through it and then

jump in here and start doing and start

making some tissue culture plan so I was

gonna do something ludwigia repens mini

super rad and maybe a couple of others

but but here's the but I'm looking for

my M&S basil medium with vitamins

remember I was talking about

microelements I'm sorry macro-nutrients

Franken macronutrients and

micronutrients there we go I get it out

this is it this is what it is this is

Morris shaggy and Scoob basil medium

with vitamins love is like the macro and

micronutrients you need you don't need

to do anything else for most plants

carnations have certain requirements

roses have other requirements but the

main thing is I left this out of my

fridge for like a month and it is toast

it's gone this stuff has to be

refrigerated it contains amino acids

amino acids are actually organic meaning

they're they're not cold chemicals you

know they will die they will degrade and

they have to be kept cold and I messed

up so I had to rush out and spend money

on getting some replacements and I gave

it actually gave me an opportunity to

stock up on more phyto hormones because

I was low let me show you guys what we

got without bashing my phone into my

face my stupid stupid face okay so which

is around

all right here we go this is how this

stuff is how we rock is how I'm rocking

as you can tell this stuff you must keep

this stuff cold cold cold show you some

of the hormones we typically work with

this is thigh diazinon solution tdz this

is one of the only man-made Fido

hormones that I will work with

I don't like artificial vital hormones

because there's they're always downsides

to them this is this is an exception

though this is a weird one I will tell

you a little history about this hey you

guys have seen my my channel my new

banner I put up there right right

bringing art nature and science together

right where our nature inside to meet

science this one little sigh history for

your tdz ideas wrong this is actually

not just a cytokinin not just a phyto

hormone this is a hardcore herbicide

this was actually developed as an

herbicide to kill dandelions and other

plants known as dicots dicots are plants

like dandelions and dandelions buddies

they're homies they always chill out

with their very annoying plants they're

hard to get rid of they put out deep

roots suckers and all kinds of things

all it takes is a little tiny fragment

one tiny fragment of a root to remain

behind after you pulled it out you're

done you still have problems so this was

actually produced to kill dicots what it

does is it actually causes the meristem

regions to meristematic regions of the

plant the meristem czar have you ever

seen a plant no grows up like a stem

plant in your aquarium chop you give it

the chop well what everyone else chopped

off what happens right you get a branch

that way and a branch that way where are

those two branches are coming off those

plant those are called meristems

and meristems are very very special

cells they're Parekh of their

parenchymal cells they can can kind of

spring into action anytime you need them

to this tdz was designed to kill dicots

in that it basically causes the plants

meristematic regions to explode that's

right it causes their meristem their

meristem cells to essentially reproduce

so quickly that the plant just explodes

and dies it causes the organism to

expire so this used in very very very

small amounts tdz is very very useful

this is naa this is like this is the

McDonald's of plant growth hormones

naphthalene acetic acid we'll go back

and look at this stuff on my chart go

back this is this is actually another

really really common one it's very

important indole-3 acetic acid you

notice this one is in a like light proof

envelope right like light can't get in

it why what do you think that is well

I'll tell you why it says right there

lights protect from light that's why

it's sealed in here now with that said a

little trivia for you guys I'm gonna set

this down last one I got is benzoyl

amino purine six good old six b.a.p

grows everything that you need it to of

course some marat/sade and Scoob these

are our micronutrients this is the news

portal place the stuff I've ruined now

trivia for you guys if I have a fight Oh

hormone in here right that is killer at

growing plants at making all kinds of

magical stuff happen right but it's so

so so so sensitive to light - right it's

photosensitive photosensitive in that

leaving it out in full spectrum light

for a full

de dum it's gone it is history that's

how sensitive it is so my question to

you is how do you think we're gonna use

that in plant tissue culture right and

we are gonna use that by the way in

plant tissue culture how are we gonna

use that stuff if it's so friggin

sensitive

you can't even whoops you can't even put

it you can't even put it underneath a

light how can it be used in plant tissue

culture plants which need light by the

way if it can't even have light

contacting it that's the trivia for you

guys I don't think you're gonna be able

to google it either I'll look at chat

here and maybe four or five more minutes

and we'll see what you guys come up with

I don't know why I put it there it

actually doesn't go there

certain guys and this is essentially a

incubation rat actually this whole rack

is it's got these lights installed on it

special LED lights that are specifically

measured down are actually selected to

give me the the right level of light for

a culture meristematic tissue cultures

so let's go back and see if you guys

know how we would deal with our weird

weird weird problem where we're using

you know a plant growth hormone that

actually can't have light contacting it

because it'll basically destroy it

long before our tissue culture is

finished how do you think we're able to

use that clearly we need to use light

right lights gonna be contacting the

tissue culture container what do you

think we do let's go back and see sorry

for the sorry for everything the audio

and everything see if we can get this

what the hell

there we go

okay we're back

all right
I am scanned up and back in chats let's

see what's going on here okay it looks

like Roxanna is jumping on it Roxana mix

it with something else

yes Roxana zonda what do you think what

do you think we mix it with Roxanna the

hint is in that PowerPoint presentation

remember almost like yeah we use this we

use that we use this we use a carbon

source the ganster Roxana you actually

got it right on fire put a blindfold on

it no Roxana don't be silly

Roxana got it would die well it's

actually not die but we use charcoal

charcoal is very beneficial it is a

additional carbon source it is also very

very good guys never bought a tissue

culture cup before and the plants lucky

and all and you look at the gel and

you're like Jill looks like brown like

it's really brown and there's like

there's like some liquid in there looks

kind of brownish yellow liquid but the

plants are fine I guess they're they're

fine yeah and you're right they're fine

those are metabolites charcoal is useful

for preventing plants from creating them

it's also useful in helping to absorb

them it's also super useful for mixing

into your medium and it turns it kind of

really dark and black and light doesn't

penetrate very far at all into that into

that medium so Roxana with the big brain

he's got it what is going on in here

faerie farts what are you guys talking

about what has been going on while I've

been working on science father fish have

you not been keeping the young ones

behaved

I'm just gonna have to redo this a

little bit here and see what's going on

a fairy forest and what's going on

Chattanooga edy put it in the gel yeah

guys are definitely and on two-on-two

all kinds of science father fish is in

trouble now he's okay well it everybody

can have fun here

that's what rep ditch blue foot but red

fish blue fish is all about I can't even

pronounce my own channels name tonight

it's a good thing we're not actually

doing tissue cultures tonight however I

do want to touch on something every

Friday at 7:30 p.m. Pacific time my time

10:30 p.m. Eastern time for a lot of you

guys I go live I will not be able to go

live nice Friday I got roped into work

and I'm on this massive project this

architectural project and I'm gonna be

up to here and work next Friday so a

week from today I will not be doing the

live stream sadly but I'm going to try

to put together a video then I will set

to premiere at that time as it may be as

a as I'm sorry it's kind of a

consolation prize we're really sorry

guys I mean we we just kind of started

this channel these live streams and I'm

already calling in sick on yeah but the

good news is while I am NOT going to be

live-streaming next Friday the Friday

after that we are going to do I'm gonna

do a three hour livestream event where

we are going to do all of this in the

lab we're gonna start the live stream

and we're gonna get right on it no chat

no messing around we'll have the audio

fixed by then we'll probably be

live-streaming directly from the YouTube

mobile app on my phone that's how I

screamed last time and the audio was way

better

so no live stream next Friday but the

Friday after that we're starting at 7:30

p.m. and we're gonna go until we're done

we're not gonna quit until we're

finished tissue tissue culturing at

least one plant from start to finish

all right and I hope all you guys will

show up and cheer me on and pick on each

other and definitely definitely have a

good time that's what this is all about

just having a good time

Roxana says in two weeks I will have

forgotten at all that's okay that's okay

we're gonna we're going to continue this

really as long as you understand a few

core concepts we touched on ex plants

are pieces of plant right we turn them

into tissue cultures until tissue

cultures reach stage four and they're

rooted technically they're not plants

they are missing some organs right we

talked about augs ins versus cytokines

held it you know they're always used in

different ratios to get our ex plant to

do certain things one really cool thing

that we will do one of these days is

will do a callus tissue culture okay you

remember a little chat that little chart

that I showed you guys and like five

stages the middle of callus fast

development that's where your office and

your cytokinin are pretty much balanced

out now when you have that sort of

scenario