Session 2 Culturing Bacteria Part 1 Plating on to agar plates
Summary
TLDRThis educational video demonstrates a microbiology lab procedure for identifying bacteria from a swab sample. The process involves inoculating the swab onto an agar plate, purifying colonies, and identifying bacteria using standard techniques. The video guides viewers through labeling agar plates, sterilizing loops, and streaking bacteria for isolation. It emphasizes the importance of proper technique to prevent contamination and ensure accurate bacterial identification, concluding with the incubation of plates for analysis.
Takeaways
- π¬ The exercise involves taking a swab sample, inoculating it onto an agar plate, and identifying the bacteria present.
- πΏ The process mimics what happens in a microbiology lab, with agar types varying depending on the specimen.
- π Clear labeling of agar plates is crucial to avoid confusion between samples.
- π₯ Sterilizing the inoculation loop before and after use is essential to prevent contamination.
- π¨βπ¬ The script describes a practical demonstration using a swab from a hypothetical patient named Mr. Smith with a post-operative infection.
- 𧫠Muller Hinton agar is used for general purposes and can grow a wide variety of bacteria.
- π Including the date on agar plates helps track the age of the sample and ensures accurate record-keeping.
- π The swab must be thoroughly rolled on the agar to ensure all bacteria are transferred.
- π― Streak plating technique is used to spread bacteria across the agar plate, starting with a primary inoculum and followed by secondary streaks to isolate single colonies.
- β± The agar plate is incubated at 37 degrees Celsius for 16 to 24 hours to allow bacterial growth before analysis.
Q & A
What is the purpose of taking a swab sample in microbiology?
-The purpose of taking a swab sample in microbiology is to collect bacteria from a specific area, such as a wound or surface, to analyze and identify the types of bacteria present.
What is an agar plate and why is it used?
-An agar plate is a type of Petri dish filled with a gelatinous substance made from agar, which provides a nutrient-rich environment for bacteria to grow. It is used to culture and isolate bacteria from a swab sample.
How does the process of purifying bacteria from a swab sample work?
-The process involves transferring bacteria from the swab onto an agar plate, allowing them to grow into colonies, then selecting individual colonies and transferring them to new agar plates to ensure a pure culture.
What types of agar are used for different specimens?
-Different types of agar are used depending on the specimen and the bacteria being cultured. For general purposes, Muller Hinton agar is used, but other types like Blood agar or MacConkey agar may be used for specific bacteria.
Why is it important to label agar plates clearly?
-Labeling agar plates clearly is crucial to avoid confusion between samples from different patients or sources, ensuring accurate tracking and identification of bacterial cultures.
What is the significance of the Universal Medical Registration Number (URN) mentioned in the script?
-The URN is used to uniquely identify patients, ensuring that samples and test results are correctly associated with the right individual.
Why is it necessary to sterilize the loop before inoculating the agar plate?
-Sterilizing the loop prevents contamination of the agar plate with unwanted bacteria or other microorganisms, ensuring that only the bacteria from the swab are cultured.
What is the purpose of rolling the swab on the agar plate?
-Rolling the swab ensures that the bacteria are evenly distributed across the agar surface, increasing the chances of growing a diverse range of bacterial colonies.
How does the streaking method help in isolating pure bacterial colonies?
-Streaking involves making lines across the agar plate with a loop, diluting the bacteria with each pass. This method helps to separate individual bacteria, allowing for the growth of isolated colonies that can be picked for purification.
What is the significance of incubating the agar plate at 37 degrees Celsius for 16 to 24 hours?
-Incubation at 37 degrees Celsius mimics human body temperature and provides optimal conditions for bacterial growth. The 16 to 24-hour period allows enough time for bacteria to multiply and form visible colonies.
Outlines
π¬ Microbiology Lab Exercise Overview
The speaker introduces a microbiology lab exercise involving the collection of a swab sample, inoculation onto an agar plate, and identification of bacteria present. The process includes purifying the bacteria to ensure a pure colony and using standard techniques for identification. The sample in focus is a 'ruined swab,' which could be replaced with any other type, such as from a bench or food. The agar type varies depending on the specimen, but the overall process remains consistent. The exercise begins with plating out the bacteria from the swab onto an agar plate, followed by purifying colonies and identifying the bacteria. The speaker humorously notes that 'Mr. Smith' is a common name associated with illness, especially in university settings, and the sample is from a post-operative infection of a patient named Mr. Smith.
π Labeling and Sterilizing in Microbiology
The speaker emphasizes the importance of labeling agar plates with the patient's name and a unique identifier, such as a universal medical registration number (URN), to avoid confusion between samples. The date is also crucial for tracking the age of the sample. The process then moves to sterilizing the loops using a Bunsen burner, ensuring they are not too hot to avoid damaging the agar or killing bacteria. The swab sample, taken from Mr. Smith's post-operative wound, is then carefully applied to the agar plate, ensuring the entire swab is used by rolling it across the surface. The speaker provides a detailed explanation of the technique, highlighting the need for thorough and careful handling to ensure accurate bacterial growth and isolation.
π‘οΈ Inoculation and Streak Plate Technique
The speaker describes the process of inoculating the agar plate with the swab sample, starting with a primary inoculum by spreading the bacteria across a portion of the plate. The streak plate technique is then demonstrated, which involves making multiple lines across the plate to dilute the bacterial concentration progressively. The aim is to achieve single colonies by the end of the streaking process. The speaker explains the importance of not reusing the loop without sterilization and not going back into the previous inoculum to prevent cross-contamination. The final step involves a 'squiggle' streak from the last streak line to further dilute the bacteria. The plate is then inverted to prevent contamination from the environment and incubated at 37 degrees Celsius for 16 to 24 hours to allow bacterial growth.
Mindmap
Keywords
π‘swab sample
π‘inoculate
π‘agar plate
π‘purify
π‘colony
π‘microbiology laboratory
π‘transport medium
π‘Bunsen burner
π‘primary inoculum
π‘streaking
π‘incubate
Highlights
Introduction to the exercise of taking a swab sample and inoculating it onto an agar plate to identify bacteria present.
Process of purifying bacteria to ensure a pure colony for accurate identification.
Overview of the steps involved in a microbiology laboratory for analyzing swab samples.
Use of different types of agar based on the specimen for bacterial growth.
Labeling of agar plates with patient details and date for clear identification.
Sterilization of loops to prevent contamination of agar plates and bacteria.
Technique of inoculating the swab sample onto the agar plate for bacterial growth.
Importance of using the entire swab to ensure all bacteria are transferred to the agar.
Spreading bacteria across the agar plate to create a primary inoculum.
Streaking method for diluting bacteria to isolate single colonies.
Turning the agar plate during streaking to facilitate the process.
Final streaking technique to isolate single bacteria colonies for identification.
Avoiding contact with previous inoculum to prevent cross-contamination.
Incubation of the agar plate at 37 degrees Celsius for bacterial growth.
Investigation of bacterial growth on plates after incubation.
Use of Muller Hinton agar plate for general bacterial growth.
Humorous mention of 'Mr. Smith' being a common name for patients with infections.
Description of transport medium in the swab tube for preserving bacteria.
Transcripts
so for this next exercise we're actually
going to take a swab sample and we're
going to inoculate that onto agar plate
and look at what sort of bacteria are
present on that we're then going to take
those the bacteria that are present
we're going to then go along and purify
them make sure that we actually have a
pure colony of bacteria and then we're
going to identify what the bacteria are
so this is really a snapshot of what
actually happens in a microbiology
laboratory now this particular sample
that we're going to be looking at is a
ruined swab but it could easily be a
swab from a bench or a food a bit of
food or whatever the the process that we
follow are all based more or less
identical just tends to be that the
types of agar that we use will vary from
from specimen to specimen so so we'll
first of all get stuck into plating out
we will start off by plating out we will
this particular process will allow us to
take the bacteria that are present on
the swab and put them onto an agar plate
the next step that we will do will be
looking at purifying or picking pure
colonies so if we have more than one
colony present we can actually take each
of the different types of colonies put
them onto another agar plate and make
sure that the organism that we're
looking at is is pure and then from
there we will go on and identify the
bacteria using some very standard
techniques so first but first of all
we'll start off by inoculating the plate
we have here a swab from a mr. Smith if
your surname is Smith I have a bad news
you
you work if you are sick well this myth
seems to be the most common name for
people being sick particularly at
university classes if it's are usually
mr. Smith or mr. Smith
they tend to be the most sick people so
today we're actually going to
investigate mr. Smith who has a
post-operative infection
mr. Smith went into hospital for a
appendectomies and has now ended up with
a post post impact post operation
infection in the wound and so we we have
a swab here from the wound so we have
here the swab from mr. Smith now this
swab is basically the same as what we've
seen before is just a sterile cotton
swab but it is in a tube which contains
maintenance me media or transport medium
now this medium is just as basically
just an agar with some butters inside
the agar to allow the bacteria to
survive but not not grow so we have our
swab from mr. Smith we now have to
culture this now to culture the swab the
first thing we need to do is we need to
find our agar plate the agar plate that
we're going to use today is a Muller
hinton agar plate this is a general a
plate for general purposes the Muller
hinton agar plate will allow us to grow
a large wide variety of different
bacteria and so we're just going to to
put this onto the agar plate and then
spread it to allow the bacteria to grow
first thing we need to do though is we
need to label the plates
and it's always very very important that
we when that we label plates and we
label them very clearly because if we do
not label them clearly we could we end
up in a situation where you have several
different plates from different samples
and you may find that you can't tell
which one belongs to which so we need to
first of all label the plate now we're
going to label the plate with the name
of the patient so we'll call it Smith it
has what a number here which is the UM
RN it's the universal medical
registration number which everybody gets
when they go into a hospital and so what
we're going to do is we'll just put the
euro RM n number but instead of writing
the entire URM n will just write the
actual numbers which is one two three
four five six nice and easy
t so we've just used that URM n the
other thing that we're going to put onto
this agar plate is the date and it's
always very important to include the
date on the agar so that we know how old
it is it's very easy for plates to to
get mixed up so if you have the dates
and you'll know approximately when the
sample was was originally set up so we
now have our plates the next thing we
need to do is start our Bunsen burner
again so the first thing we need to do
is we need to sterilize the loops so
first of all we put that into the flame
and once it goes orange come out all the
way down all the way back up
and we put that to the side once again
take our loop let it warm up go down and
then up again and that's nice and
sterile now it's very important when we
do this that we don't allow don't use a
hot loop and the reason for that is if
we use a hot loop and we put that on to
our agar it will actually basically melt
the agar and it will also kill any
bacteria that happen to be on the agar
so it's always very very important to
make sure that you sterilize the loop
and then allow it to stick I only need
to sit for about 10-15 seconds that will
be enough for it to actually to stop to
get cool enough and be ready for the
next stage so we have our labelled plate
the next thing we need to do is we need
to add our now swab sample onto the
plate so we just take the swab out and
just put a little bit onto the surface
now as you can see I'm actually as I'm
putting it onto the agar I'm also just
swiveling it back and forth so it goes
so that we're actually using all the
swab the reason for that is that when
you take a swab theoretically you're
supposed to do you're supposed to use
the entire swab but quite often you'll
get people that will just go up to swab
the surface on one side and if you
happen to to use the other side when
you're actually putting it onto the agar
then you'll end up with no bacteria so
it's very important to make sure that
you roll the swab as you're actually
putting it on to the surface
okay so the next thing that we're going
to do is we're going to spread the egg
that the bacteria across this plate if
you actually turn to Appendix 1 unless
you find there is a detailed description
of how to do this but it's quite a
simple process the first thing that I'll
do is using one end of the loop you can
see here that I'm actually going to use
the bottom of the loop so you've got the
bottom and the top so I'm going to use
this bottom section of the loop and I'm
going to spread the bacteria across the
plate now this is what we call our
primary inoculum and we just basically
take the swab that we've added that we
put on and then just spread that across
let's go back and forth and use about
you know a quarter or a little bit less
than a quarter of a plate to spread that
into primary inoculum now what i'm going
to do next is i'm actually going to turn
that loop over so i've started on on
this side i'm going to turn that over
so from on to the second side and then
I'm going to streak so all I'm going to
do is I'm going to take this from the
primary inoculum and put a line across
and then another line and another line
another line and usually you do four or
five of those okay next I'm going to
take this loop I'm going to put that
down and I'm going to inoculate and then
sterilize the loop
okay well when I'm actually putting the
agar down you'll notice that I'm
actually putting it so that the agar is
up so basically what you think is the
bottom of the plate is actually going up
so we always make sure that we when we
inoculate when we're using agar plate
that we put basically the face down and
the reason for that is that it stops
bacteria from being able to to fall onto
the plate from the atmosphere if we were
to just leave this plate sitting like
this we would have spores of fungus
we have bacteria and so forth all just
growing dropping onto the plate which
will then allow it to grow so we always
put it upside down we take our second
loop and
we from this we take out from our
primary inoculant to our second we call
this now what we strike streaked out
before the secondary inoculum we just go
one two three four and then we turn the
loop now you'll notice as I do that I'm
also also turn the plate so we've gone
from inoculating on this side to that
side and I just turned the plate so it
makes it easier for me to do the next
thing makes a lot of streaks just go one
two three I always do
5 4 3 no exact correct way of doing
these when when you played out different
microbiologists played out differently
but I've found that 5 4 3 works best for
me
the last streak that we do is we take
our loop go through this the last
streaking out that we did and we take
out from there and then just squiggle
the the loop back and forth down the
middle of the plate now it's very
important when you're doing this that
you don't go back into the previous
inoculum so on here we do one streak out
and then make sure that we don't touch
this end here and more importantly that
we don't touch this side here and the
reason for that is that as the primary
inoculum which will have the most amount
of bacteria now the theory behind this
is what we're doing is we're diluting
out the number of bacteria each time
that we scrape across the surface so we
started off with with probably lots of
bacteria here and each time that we
streak out
we're ending up with less and less
bacteria so and the aim of this is to
get single colonies so from here what we
will do is we will incubate this plate
at 37 degrees Celsius for 16 to 24 hours
we just usually say we take them out
overnight and and then we will
investigate what is actually growing on
these plates next
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