Session 2 Culturing Bacteria Part 1 Plating on to agar plates

Summary

This 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

• 🔬 The exercise involves taking a swab sample, inoculating it onto an agar plate, and identifying the bacteria present.
• 🌿 The process mimics what happens in a microbiology lab, with agar types varying depending on the specimen.
• 📝 Clear labeling of agar plates is crucial to avoid confusion between samples.
• 🔥 Sterilizing the inoculation loop before and after use is essential to prevent contamination.
• 👨‍🔬 The script describes a practical demonstration using a swab from a hypothetical patient named Mr. Smith with a post-operative infection.
• 🧫 Muller Hinton agar is used for general purposes and can grow a wide variety of bacteria.
• 📅 Including the date on agar plates helps track the age of the sample and ensures accurate record-keeping.
• 🔁 The swab must be thoroughly rolled on the agar to ensure all bacteria are transferred.
• 🎯 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.
• ⏱ 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

00:00

🔬 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.

05:02

📝 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.

10:03

🌡️ 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.

Keywords

💡swab sample

A swab sample refers to a method of collecting a small amount of biological material, such as bacteria, from a specific area. In the context of the video, a swab sample is taken from a patient named Mr. Smith who has a post-operative infection. The sample is then used to inoculate an agar plate to identify the bacteria present.

💡inoculate

Inoculation in microbiology involves transferring a small amount of a microorganism to a culture medium to grow and study it. In the video, the swab sample is inoculated onto an agar plate to allow the bacteria to grow and be observed.

💡agar plate

An agar plate is a type of petri dish filled with agar, a gelatinous substance that provides a surface on which microorganisms can grow. It is used in the video to culture the bacteria from the swab sample. The specific type mentioned is Muller Hinton agar, which is a general-purpose medium for growing a wide variety of bacteria.

💡purify

Purifying bacteria in a microbiology lab involves isolating a single strain or colony to ensure that the study focuses on a pure culture. The video mentions purifying the bacteria from the agar plate to obtain a pure colony for further identification.

💡colony

A colony in microbiology is a visible group of identical microorganisms that have grown from a single cell or group of cells. The video discusses picking pure colonies from the agar plate to ensure that the bacteria being studied are not mixed with other types.

💡microbiology laboratory

A microbiology laboratory is a facility where the study of microorganisms such as bacteria, viruses, and fungi is conducted. The video provides a snapshot of the procedures that occur in such a laboratory, including the handling and analysis of swab samples.

💡transport medium

Transport medium is a substance used to preserve the viability of microorganisms during their transport from the collection site to the laboratory. The video mentions that the swab is in a tube containing transport medium to keep bacteria alive but not growing.

💡Bunsen burner

A Bunsen burner is a type of gas burner used in laboratories to produce a single flame, often for heating or sterilization. In the video, a Bunsen burner is used to sterilize the inoculation loop before it is used to transfer bacteria to the agar plate.

💡primary inoculum

The primary inoculum is the initial transfer of bacteria from the swab to the agar plate. The video describes spreading the bacteria from the swab across a portion of the agar plate to create the primary inoculum.

💡streaking

Streaking is a technique used to isolate single colonies of bacteria by spreading them across the surface of an agar plate in a series of lines. The video demonstrates streaking from the primary inoculum to create a secondary inoculum and eventually to isolate single colonies.

💡incubate

Incubation in microbiology involves keeping the culture at a specific temperature and condition to allow the bacteria to grow. The video mentions incubating the agar plate at 37 degrees Celsius for 16 to 24 hours to allow the bacteria to grow before analysis.

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