Subculturing Techniques

CULTURING MICROBES:
SUB-CULTURING TECHNIQUES
Biotechnology Protocols
41
Subculture Techniques
SUBCULTURING MICRO-ORGANISMS
Source: NQ Curriculum Support Intermediate 2 Biotechnology
(Unit 2 Student Materials)
In the laboratory, micro-organisms are usually grown or cultured in liquid
medium (broth) or on solid medium (agar plates or slopes). Growth of
bacteria and yeasts shows as cloudiness or turbidity in the broth although
sometimes bacteria grow as a layer on the surface of the broth or at the
bottom of the culture tube.
The growth on plates depends upon how the plate has been inoculated.
Types of inoculations on agar plates

Subculturing is the aseptic transfer of micro-organisms from a culture to
fresh medium. The freshly inoculated medium is then incubated at the
temperature appropriate for growing the organism.
There are four subculturing procedures with which you should become
familiar. They are:
Solid to solid: the transfer of bacteria or fungi from an agar slope or plate
culture to an agar plate
Solid to liquid: the transfer of bacteria or fungi from an agar slope or plate
culture to a broth
Liquid to solid: the transfer of bacteria or fungi from a broth culture to an
agar slope or plate
Liquid to liquid: the transfer of bacteria or fungi from a broth culture to a
broth
Containers of culture media to be inoculated must be labelled with initials,

date and name of organism. To prevent possible confusion, plates are
marked on the underside while tubes and bottles must be labelled on the side.
Lids are not labelled.
42
The use of aseptic technique minimises the risk of contamination of cultures

and also reduces the risk of micro-organisms from the laboratory cultures
escaping to the environment.
You will use a wire loop, a straight wire, forceps or a scalpel to subculture
bacteria or fungi or to transfer specimens to a slide. The wire loop is the basic
tool of the microbiologist and you should learn to handle it correctly.
Flame Sterilisation of Instruments
Metal instruments used to transfer micro-organisms are sterilised by heating
till red hot in the blue part of the Bunsen flame heat. They must be heated till
red hot to make sure that any contaminating bacterial spores are destroyed &
this process must be carried out before and after use.
Good flaming technique is very important to avoid contamination of the
surrounding air with aerosols.
A Bunsen burner is lit and the air hole opened fully to provide a blue flame.
The operator holds the loop between thumb and fingers as if holding a pencil
very loosely, at an angle that is almost vertical.
43
The loop is placed in the light blue cone of the flame. Positioning the loop in
this cool area of the flame allows any liquid to dry out and prevents formation
of aerosols. Aerosols are fine liquid or solid particles that are dispersed into
the air and might contain micro-organisms.
Placing a loop in blue cone of flame

After any liquid material has evaporated, the loop is drawn slowly up into the
hottest region of the flame (immediately above the light blue cone) and held
there until it is red hot.
Drawing loop into hottest region of flame

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The loop is then withdrawn from the flame and allowed to cool before touching
micro-organisms.
Cooling the loop

Straight wires may be sterilised in the same way.
Aseptic Transfer Operations
Once the loop has been sterilised and cooled, it is used to remove organisms
(the inoculum) from a culture and inoculate sterile growth medium. When
working with micro-organisms, aseptic techniques are used to avoid escape of
organisms to the environment.
To minimise the chances of contamination, cultures and media are exposed to
the air for the minimum time it takes to perform a manipulation or to make
observations. All subculturing procedures are carried out close to a Bunsen
flame.
After flaming, the loop is not put down until the procedure has been
completed.
Lids of Petri dishes are never completely removed. They are opened just
enough to allow entry of the loop to perform the manipulation and minimise
exposure to the air.
Liquid cultures are disturbed as little as possible to reduce the risk of aerosol
formation. Lids from cultures are never placed on the bench surface
where contamination might occur. They are removed from the bottle or tube
using the little finger, held there while manipulation of the culture takes place
and then replaced. The rest of the hand is free to carry out the manipulation.
Lids are tightened to prevent spillage before incubation.
45
To remove any contaminating organisms on the neck of a bottle, it is passed

through a hot Bunsen flame on removal and before replacement of the lid.
The loop must be flamed to red heat when the subculturing procedure is
finished.
The table below summarises potential points of contamination and the
techniques employed to minimise the risk.
Contamination Risk
Precaution
Inoculating loop
Flame and cool.

Do not lay down loop until procedure
is complete.
Work close to Bunsen flame.
Opening of Petri dish (solid medium)
Open lid for as short a time as

possible.
Open lid just enough to insert wire
loop.
Opening of liquid cultures (bottles,

tubes)
Hold the lid in crook of little finger

never place on work bench.
Pass neck through a hot Bunsen
flame before insertion and after
withdrawal of loop to kill any
contaminating organisms.
Incubation of cultures
After inoculation, cultures are incubated at a given temperature. Petri dishes
should be placed upside down to prevent condensation dropping on to
cultures. The lids of Petri dishes should be secured to the base with diagonal
strips of sellotape. Bottles should be stored upright in a container which will
prevent them being knocked over.
Subculture results
Subculturing has been successful when the transferred organisms have
grown in the new medium without contamination.
Contaminating organisms can usually be observed on plate cultures as single
colonies with a different appearance. In a broth culture, it is impossible to tell
by simply observing the broth whether the culture is pure. The liquid culture
must be streaked out on an agar plate to determine if there are any
contaminants.
In the practical work associated with this course, you will generally be working
with known organisms and with pure cultures. A pure culture is one in
which there is only one type of organism.
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STREAK PLATES
When plating out micro-organisms various techniques can be used.
However, when attempting to obtain isolated colonies the streak-plate
method is routinely used.
Streak Plate Inoculation: Solid to Solid
Instructions
These instructions are for right-handed people. If you are left handed, please
reverse the instructions accordingly.
1 Wear a lab coat.
2 Prepare your work space on the bench, collect the materials and set them
out correctly on the bench.
3 Label the bases of the Petri dishes containing sterile nutrient agar with
initials, date, name of micro-organism and S
S (for solid-to-solid
transfer).
4 Hold the loop in the right hand.
5 Flame the loop and allow to cool.
Do not put down loop or wave it around.
6 Lift the lid a little of the Petri dish containing the inoculum with the left
hand.
7 Touch a single colony with the wire loop.
8 Withdraw loop. Do not put down loop or wave it around!
9 Replace lid of Petri dish.
10 Partially lift the lid of the Petri dish containing the solid medium.
47
11
Holding the loop parallel with the

surface of the agar, smear the inoculum
backwards and forwards across a small
area of the medium. Replace lid.
12
Flame loop and allow to cool.
13
Turn plate. Streak loop from A across

the surface of the agar in three parallel
lines. Replace lid.
14
Flame the loop and allow to cool.
15
Turn plate. Streak loop from B across

the surface of the agar in three parallel
lines. Replace lid.
16
Flame loop and allow to cool
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17 Turn plate. Streak loop from C to D across

the surface of the agar as shown, without
touching area A.
18 Replace lid of Petri dish

19 Flame loop
20 Incubate plate upside down at 30C for 72
hours
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Streak Plate Inoculation: Liquid to Solid

Instructions
These instructions are for right-handed people. If you are left handed, please
reverse the instructions accordingly.
1 Wear lab coat and eye protection.
2 Prepare your work space on the bench, collect the materials and set them
out correctly on the bench.
3 Label the underside of plates with initials, date name of micro-organism
and L
S (for liquid-to-solid transfer).
4 Turn the plate upright.
5 Loosen the tops of the universals containing the broth cultures so that they
can be removed easily.
7 Flame the loop and allow to cool. Do not put down loop or wave it around.
8 Lift the universal containing the inoculum with the left hand.
9 Remove the lid of the universal with the right hand. Do not put down the
lid.
10 Flame neck of universal.
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11 Insert the loop into the culture (bring bottle to loop, not loop to bottle) and
withdraw. Take care not to touch the side of the bottle or its mouth.
12 Flame the neck of the universal.
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13 Replace the lid on the universal using the little finger. Turn bottle, not lid.
14 Place the universal on the bench.
15 With the left hand, partially lift the lid of the Petri dish containing the solid
medium.
16
Holding the loop parallel with the

surface of the agar, smear the
inoculum backwards and forwards
across a small area of the medium.
Replace lid.
17
18
Turn plate. Streak loop from A

across the surface of the agar in
three parallel lines. Replace lid.
19
Flame the loop and allow to cool.
52
20
Turn plate. Streak loop from B

across the surface of the agar in
three parallel lines. Replace lid.
21
22
Turn plate. Streak loop from C to D

across the surface of the agar as
shown, without touching area A.

24 Flame loop.
25 Incubate plate upside down at 30C for 72 hours.
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SMEAR PLATE: SOLID TO SOLID

Source: HSDU Biology and Biotechnology Microbiological Techniques
Intermediate 1- Advanced Higher Folder
2 Flame the loop and allow to cool. Do not put down loop.
3 With the left hand, lift the lid a little of the Petri dish containing the
inoculum.
5 Withdraw loop carefully without touching plate. Do not put down loop or
wave it around.
7 Partially lift the lid of the agar plate and gently rub the loop across the
surface of the agar (see diagram below). Take care not to break the agar
surface.

9 Sellotape lid to base of plate (two diametrically opposed pieces).
10 Invert and incubate under appropriate conditions.
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SMEAR PLATE: LIQUID TO SOLID

5 Remove the lid of the universal with the little finger of the right hand. (Turn
the bottle, not the lid). Do not put down the lid.
7 Bringing the bottle to the loop, insert the loop into the culture broth and
withdraw.
8 At all times, hold the loop as still as possible.
10 Replace the lid on the universal using the little finger, turning the bottle not
the lid.
11 Place universal on bench.
12 Partially lift the lid of the agar plate and gently rub the swab across the
surface of the agar (see diagram below). Take care not to break the agar
surface.

14 Sellotape lid to base of plate (two diametrically opposed pieces).
15 Invert & incubate under appropriate conditions.
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INOCULATING A LIQUID CULTURE: SOLID TO LIQUID

2 Flame the loop and allow to cool. Do not put down loop.
3 With the left hand, lift the lid a little of the Petri dish containing the
inoculum.
5 Withdraw loop carefully without touching plate. Do not put down loop or
wave it around.
7 Lift a universal of sterile nutrient broth in the left hand.
8 Remove the lid of the universal with the little finger of the right hand which
still holds the charged loop. Do not put down the lid.
10 Insert the loop charged with inoculum into the sterile broth. Touch on the
inside of the universal and withdraw.
11 Flame the neck of the universal, replace lid and place the universal on the
bench.
12 Flame the loop and place on heat resistant mat.
13 Tighten lid of universal to make secure but do not overtighten.
14 Incubate under appropriate conditions.
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INOCULATING A LIQUID CULTURE: LIQUID TO LIQUID

1 Loosen the tops of universals containing the broth cultures so that they
7 Bringing the bottle to the loop, insert the loop into the culture broth and
withdraw.
8 At all times, hold the loop as still as possible.
the lid.
still holds the charged loop. Do not put down the lid.
15 Insert the loop charged with inoculum into the sterile broth. Touch on the
inside of the universal and withdraw.
bench.
17 Flame the loop and place on heat resistant mat.
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FROM LIQUID TO LIQUID USING A PASTEUR PIPETTE

2 Remove the pipette from its sterile container taking care not to touch the
tip, attach bulb and hold in the right hand.
6 Squeeze the teat of the pipette before it enters the broth so that it does not
cause bubbles and possible aerosols and withdraw a little of the culture.
7 At all times, hold the pipette as still as possible.
the lid.
Take care that the culture does not drip from the pipette.
still holds the pipette. Do not put down the lid.
14 Insert the pipette into the universal & gently release the required number of
drops from the pipette into the culture and withdraw pipette.
Do not agitate or cause bubbles in the liquid medium.
bench.
16 Place pipette in discard jar.
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SPREAD PLATE
Spread plates, also known as lawn plates, should result in a culture spread
evenly over the surface of the growth medium. This means that they can be
used to test the sensitivity of bacteria to many antimicrobial substances, for
example mouthwashes, disinfectants and antibiotics.
1 Loosen the lid of the bottle containing the broth culture.
2 Hold a sterile pipette in the right hand and the bottle/test tube containing
the broth culture in the left.
3 Remove the lid/plug of the bottle/test tube with the little finger of the right
hand and flame the neck.
4 With the pipette, remove a small amount of broth.
5 Flame the neck of the bottle/test tube and replace the lid/plug.
6 With the left hand, partially lift the lid of the Petri dish containing the solid
nutrient medium.
7 Place a few drops of culture onto the surface [about 0.1 cm 3].
8 Replace the lid of the Petri dish.
9 Place the pipette in a discard jar.
10 Dip a glass spreader into alcohol, flame and allow the alcohol to burn off.
11 Lift the lid of the Petri dish to allow entry of spreader.
12 Place the spreader on the surface of the inoculated agar and, rotating the
dish with the left hand move the spreader in a top-to-bottom or a side-toside motion to spread the inoculum over the surface of the agar. Make
sure the entire agar surface is covered.
This operation must be carried out quickly to minimize the risk of
contamination.
13
14
15
16
Replace the lid of the Petri dish.

Flame spreader using alcohol.
Let the inoculum dry.
Seal and incubate the plate in the inverted position.
59
SUBCULTURING MOULDS USING i) SCALPEL ii) CORK BORER OR

BLUNT END OF PASTEUR PIPETTE
1 Dip the implement in ethanol, place in Bunsen flame briefly and allow
ethanol to burn off. Do not put down.
2 Keep the ethanol at a safe distance from the Bunsen flame.
3 Lift the lid a little of the Petri dish containing the inoculum with the left
hand.
4 Hold the inoculating implement in the right hand.
5 i) with scalpel, cut a square shape in the fungal mycelium;
OR
ii) with forceps, pick up a little piece of mycelium;
OR
iii) with cork borer or pipette, cut a core of mycelium;
(if the centre of the colony is producing spores, remove the inoculum from
the edge of the colony).
6 Remove a cube, strands or core of agar with its fungal mycelium
7 Withdraw implement from Petri dish.
9 At all times, hold the inoculating implement as still as possible.
10 Partially lift the lid of the Petri dish containing the sterile malt agar medium.
11 Place the cube/bore of agar or fungal mycelium on to the centre of the
sterile agar. Use a mounted needle which has been flamed in ethanol to
assist with the transfer process if necessary.
12 Withdraw implement.
13 Dip the implement in ethanol, flame as above and place on heat resistant
mat.
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CULTURING MICROBES:

Types of inoculations on agar plates

Containers of culture media to be inoculated must be labelled with initials,

The use of aseptic technique minimises the risk of contamination of cultures

Placing a loop in blue cone of flame

Drawing loop into hottest region of flame

Cooling the loop

To remove any contaminating organisms on the neck of a bottle, it is passed

Flame and cool.

Opening of Petri dish (solid medium)

Open lid for as short a time as

Opening of liquid cultures (bottles,

Hold the lid in crook of little finger

Holding the loop parallel with the

Flame loop and allow to cool.

Turn plate. Streak loop from A across

Flame the loop and allow to cool.

Turn plate. Streak loop from B across

Flame loop and allow to cool

17 Turn plate. Streak loop from C to D across

18 Replace lid of Petri dish

Streak Plate Inoculation: Liquid to Solid

12 Flame the neck of the universal.

Holding the loop parallel with the

Flame loop and allow to cool.

Turn plate. Streak loop from A

Flame the loop and allow to cool.

Turn plate. Streak loop from B

Flame loop and allow to cool.

Turn plate. Streak loop from C to D

23 Replace lid of Petri dish.

SMEAR PLATE: SOLID TO SOLID

8 Replace lid of Petri dish.

SMEAR PLATE: LIQUID TO SOLID

13 Replace lid of Petri dish.

INOCULATING A LIQUID CULTURE: SOLID TO LIQUID

INOCULATING A LIQUID CULTURE: LIQUID TO LIQUID

FROM LIQUID TO LIQUID USING A PASTEUR PIPETTE

Replace the lid of the Petri dish.

SUBCULTURING MOULDS USING i) SCALPEL ii) CORK BORER OR

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