Culturing Bacteria

okay so

i'm making a video today

of videos these videos uh made

like six years ago and there some

students have difficulties viewing them

because of the

the you know the format they're in

you should do you should be taking notes

while you watch these videos

lots of the information is on the lab

powerpoint but not all of it

so if i mentioned something you might

want to jog it down

okay in this video we're going to i'm

going to explain how we culture bacteria

and in order to do that we need to have

a food source

for the bacteria to grow on and that's

called

media all right media needs to contain

both a carbon and energy source

and one of the more commonly used

media's in lab

is called the tsb which stands for

tryptic soy

broth right this is a

tryptic digest of a soybean extract

and if it's liquid why do you call it a

broth

right well i'll show you how to

inoculate that soon

all right and if you add agarose to the

broth it becomes

tsa because of the agar in it

right it's a solidifying agent so it

goes from uh

this liquid to a solid

it's kind of like gelatin same

consistency so it's easy to puncture it

you know when we inoculate it now when

but when they uh it's a liquid if you

before it becomes a solid if you put it

on a

angle like this you can generate what's

called a slant

and it generates a larger surface area

for the bacteria to grow on they

literally grow on the

surface of these medias

now and for many times when we do a lab

we're going to be inoculating different

biochemical tests

and we're going to ask do the bacteria

you know

use the carbon energy sources found in

the media

or are they positive for one particular

test or not

all right so this is an example of the

cinnamon citrate tube

and it's actually a slant it contains

citrate

if the bacteria are able to use citrate

as a carbon and energy source or if

they're able to

ferment citrate then they're positive

for this test

and we can tell that because when they

ferment

the the citrate the ph changes

right and most of these biochemical

tests will have ph

indicators in them in this case it's

bromfiemol

blue it appears green at this ph

right but if they are able to ferment

citrate if they have the correct enzymes

for that then the media will change

color

it will turn to a blue right so to send

in citrate to when it becomes blue

you say that they're positive for that

particular biochemical test

okay so that was

so i'm going to put two videos on this

video together

so this is the second video about

inoculating the media if you were to do

the lab

in person okay so uh now i want to

inoculate some media and show you the

proper technique for that

all right to begin with we need to start

with a sterile loop

right that's how media is inoculated we

want to transfer bacteria

from a source and then place the

bacteria

in a sterile uninoculated media

and we can use a needle

right it's just a wire straight wire or

a loop

which is a wire with a loop at the end

right normally the loop is the best to

use unless

the needle is necessary all right so i'm

going to

when we start when you turn the gas on

be sure

that the handle is pointing in the

direction of the tube the

in the direction of the hose okay

and then i'll light the flame the flame

ought to be you know roughly

four or five inches up and then place

the loop

in the center of the flame it should

turn bright red

and it takes it about 30 seconds to cool

down once you do that

all right if you go too quickly it'll

kill the bacteria that you're trying to

transfer

now the bacteria when you grab the

bacteria you don't need to grab

too many right what i normally do is

just

touch the surface and leave it nick

right if i see any bacteria on my loop

there's more than enough bacteria

there there's literally maybe 100 000 to

a million

so i want to place them into some liquid

media

okay so i remove the cap use my little

pinky

you can flame the loot the the surface

of

the tube if you like and then place the

needle

down in the tube and then

i usually twirl the loop

and give it about 10 seconds so that the

bacteria

dissolve into the liquid

okay and then i could flame the top of

the

tube put the cap back on

so now what i've done is i've added

a couple thousand sold bacteria i've

removed them from this service

and inoculated a fresh media

now if i wanted to inoculate

a a slant now i could

grab some bacteria

right say and i'm going to put these

bacteria on the surface of this slant

all right so

you start at the bottom of the slant

and then just move the slab back and

forth hold it

the needle like you would a pencil and

just work your way

up the surface of the slant and you can

usually see from the reflection where

you've left

a mark with the loop right and you still

have inoculated the surface of this

slant with bacteria

right in a day there would be plenty of

bacteria

growing there

didn't take long for them to grow now

sometimes you need to use a needle

instead of

a loop okay so i've got a needle

and i'll

sterilize it in the flame i need to give

it time to cool down

i touch the surface of the plate just to

cool it down real

and i'll just grab just a little bit a

few bacteria there

they're not that many but i don't need a

lot

like this is a deep tube

right into all i want to do now is take

the needle

and try to get into the center

[Music]

of the tube it doesn't have to be the

exact center but so i've inoculated

or stabbed this deep tube

right so bacteria expect now if this

were semi-solid media

right this would be a way of determining

if bacteria or remotely or not right

they could move away from where i've

just inoculated or stabbed the media

then

we could we could determine if they were

mortal or not if they have a flagella

for instance all right so flame the loot

needle i'm done with it now things like

the sun and citrate

require a stab

and the surface to be inoculated

right so someone described it as a

standard sweep

yesterday i thought that was a good idea

so

when i to do that then i need some

bacteria make sure this is

needle you need to use a needle for this

all right i'm going to grab some

bacteria

and stab the this is a little bit more

difficult than the deep tube

i'm going to do my best try to get

close to the center i'm stabbed

goes to the bottom i come off off of it

and now i'm doing the surface with a

needle and it's hard not to

dig into the auger do the best you can

with that if you

if you dig into the auger it's not going

to be a big problem right

but you could flame the surface

right now that's

the deep tube or the that's a cinnamon

citrate rather

now what i want to do is show you how to

say if we needed to do a

uh generate a lot of bacteria on the

surface of this

plate here right in order to do that i

could use a loop

and i want to grab some bacteria i cool

this loop down

just touch the surface and now

i want to inoculate the entire surface

here

okay so in order to do that i just start

at one end

and i work the loop back and forth

trying not to

dig into the auger

you can also use a cotton swab for this

then i'm going to turn it about 90

degrees

and then continue

to spread the bacteria as evenly as i

can across the surface

and then you would expect the bacteria

to grow in an even

lawn it's called there's no

individual colonies

okay that that's one way and then the

other method i want to describe

is when we need to make a street plate

okay and a street plate is necessary

when you start with a mixed

culture of bacteria a mixed culture of

bacteria means there's

more than one bacteria growing in a

media

and you want to separate it into a pure

culture

so in order to do that you should

remember those definitions say imagine

that i

put i've inoculated this loop with some

bacteria that

from a mixed culture and now i'm going

to do a stream plate so

when you do this method you want to

start

you want to make basically four

quadrants

right and the first one i usually make

is very small

i'm just trying to get most of the

bacteria that was on the loop

into this one quadrant i'm going to turn

the plate

i'm gonna make the second quadrant now

i'm trying to do in this process is to

reduce the amount of bacteria

that i'm spreading over the plate so the

second time

i may just go and grab some from this

area and bring it in to the second swap

right and that's called the second

quadrant and it might not be a bad idea

between quadrants to sterilize the loop

right so that when you spread it into

the next quadrant there aren't any

bacteria on the loop

what you're doing then is just grabbing

some

when i take go into this maybe twice

and make the third quadrant

now the first segment of their quadrants

are relatively the same size

the fourth quadrant i'm making i'm going

to

right i'm cooling it down all right i'm

going to make that one the biggest

so i'm going to go back into this

quadrant once twice

and then make a large streak on the rest

of the surface of the plate

okay now i'm doing that so that

when i give these

bacteria a chance to grow right there'll

be plenty of bacteria in the first and

second but

in the fourth quadrant i should be

reducing the neurobacteria to down to

one

and on that streak where there's one

bacteria you'll see just one colony

right and then you would consider that

colony of pure culture

because it was generated by from one

bacteria

okay so

right the i i'm not going to ask you

specific questions

uh about how to do these things right i

i basically made these how to do videos

for in class lab right if you're taking

this online

then you should know what a street plate

is right you should understand the

concept of a mixed culture pure culture

uh those sorts of definitions okay

you should understand auger is added to

liquid media to generate solid media

right a pure colony is a colony with

only one type of bacteria

right a mixed sculpture contains more

than one type of bacterial so those

sorts of things

all right you should be familiar with