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STREAKING

CULTURE PLATES IN
BACTERIOLOGY
Dr.T.V.Rao MD

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Dr.T.V.Rao MD

Think Before Inoculation


Before

inoculation,
important information
is written on the bottom
of the plates, close to
the rim

1date

of inoculation

2.temperature
3.duration

of incubation

of incubation

4.microorganism
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inoculated
Dr.T.V.Rao MD

What is streaking
The

most common method of inoculating an agar plate is


streaking.

Streak

plates

1.With

this method, a small amount of sample is placed on the


side of the agar plate (either with a swab, or as a drop from an
inoculating loop if the sample is a liquid).

2.A

sterile loop (flamed until red hot, then cooled by touching the
agar away from the inoculated sample) is then used to spread
the bacteria out in one direction from the initial site of
inoculation. This is done by moving the loop from side to side,
passing through the initial site
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Dr.T.V.Rao MD

What is streaking
The

loop is then sterilised (by flaming)


again and the first streaks are then
spread out themselves.

.This

is repeated 2-3 times, moving


around the agar plate.

What

should happen is that single


bacterial cells get isolated by the
streaking, and when the plate is
incubated, forming discrete colonies
that will have started from just one

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Dr.T.V.Rao MD

The essential step in inoculating


culture plates
There

are several
essential
precautions that
must be taken
during inoculation
procedures to
control the
opportunities for
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Dr.T.V.Rao MD

Prompt action with


must not be
Optimal utility
started until
Operations

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all
requirements
are within
immediate
reach and
must be
completed as

Dr.T.V.Rao MD

Expose the inoculum in test


tubes and containers for
minimal Time
Inoculum is grown in test
tubes and must be open
for the minimum amount
of time possible and
while they are open all
work must be done close
to the Bunsen burner
flame where air currents
are drawn upwards.
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Dr.T.V.Rao MD

The Neck of Test tube


On being to
containing inoculum
be
opened,
the neck of a test
heated briefly
tube or bottle

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must be
immediately
warmed by
flaming so that
any air movement
is outwards and
the vessel held as
near as possible

Dr.T.V.Rao MD

Exposure to
Environment should
limited
to
minimum
During
manipulations
involving a Petri
dish, exposure of
the sterile inner
surfaces to
contamination
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Dr.T.V.Rao MD

Work with absolute sterility


The

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parts of sterile
pipettes that will be
put into cultures or
sterile vessels must
not be touched or
allowed to come in
contact with other nonsterile surfaces, e.g.
clothing, the surface of
the working area, the
outside of test
Dr.T.V.Rao MD
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tubes/bottles

Work Using a wire loop


Wire

loops are sterilised using red


heat in a Bunsen flame before and
after use. They must be heated to
red hot to make sure that any
contaminating bacterial spores
are destroyed. The handle of the
wire loop is held close to the top,
as you would a pen, at an angle
that is almost vertical. This leaves
the little finger free to take hold
of the cotton wool plug/screw cap
of a test tube/bottle
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Dr.T.V.Rao MD

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Flaming procedure
The

flaming procedure is
designed to heat the end
of the loop gradually
because after use it will
contain culture, which
may splutter on rapid
heating with the
possibility of releasing
small particles of culture
and aerosol formation

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Dr.T.V.Rao MD

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Handling of the
Inoculating loop
Position the
handle end of
the wire in the
light blue cone
of the flame.
This is the cool
area of the flame

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Perfect the art handling


the Inoculating loop
Draw the rest of
the wire upwards
slowly up into
the hottest
region of the
flame,
(immediately

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Heat and cool the Loop

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Ensure the full


length of the wire
receives adequate
heating.
. Allow to cool
then use
immediately.

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Handle the Inoculating loop


Do not put the
loop down or wave
it around..
Resterilise the loop
immediately after
use.
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Dr.T.V.Rao MD

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Be careful some times the Loop may not


contain inoculum must redraw
the inoculum
If a loop does not hold

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any liquid the loop has


not made a complete
circle. To correct the
problem, first ensure
that the loop has been
sterilised and then
reshape the loop with
forceps. Do not use
your fingers because of
the possibility of
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puncturing the skin. 17

Working with bacteria


and yeast Streak plate

The

loop is used for


preparing a streak
plate. This involves
the progressive
dilution of an
inoculum of bacteria
or yeast over the
surface of solidified
agar medium in a
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The aim of the procedure is


to obtain single isolated
pure colonies

Loosen the cap


of the bottle
containing the
inoculum.
Hold the
loop in the right
hand.
.

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Handling matters in
getting the right inoculum
Lift the bottle/test tube
containing the inoculum
with the left hand. .
Remove the cap/cotton
wool plug of the
bottle/test tube with the
little finger of the right
hand. .
Flame the neck of
the bottle/test tube.
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Handling loop and Inoculating


the material matters
Insert

the loop into


the culture broth
and withdraw. At
all times, hold the
loop as still as
possible.
. Flame neck of the
bottle/test tube.

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Do not Practice the way

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Replace the
Practice for Perfection

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cap/cotton wool
plug on the
bottle/test tube
using the little
finger. Place
bottle/test tube on
bench. . Partially
lift the lid of the
Petri dish
containing the

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Practice , Practice best way to


Perfection
Hold the charged loop
parallel with the
surface of the agar;
smear the inoculum
backwards and
forwards across a small
area of the medium
(see streaked area .

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Dr.T.V.Rao MD

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Follow the Instructions for


safe keeping of Petri dish
Remove the
loop and close
the Petri dish.
.
Flame the loop
and allow it to
cool.

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Dr.T.V.Rao MD

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Streak the Plates


a
With thein
cooled
loop streak the
defined Manner

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plate from area


across the
surface of the
agar in three or
four parallel
lines . Make
sure that a
small amount of
culture is

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Streak the Plates in a


defined Manner

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Remove the loop and


close the Petri dish. .
Flame the loop and
allow to cool. Turn the
dish through 90
anticlockwise again and
streak from across the
surface of the agar in
three or four parallel
lines .

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Remove

Carry with further


inoculations

the loop and


close the Petri dish. .
Flame the loop and
allow to cool. Turn the
dish through
90anticlockwise and
streak loop across the
surface of the agar
from into the centre
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Remove
Complete the work
Close
the the
loop
and close the Petri
petri dish

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dish. Flame the


loop.
. Seal and
incubate the plate
in an inverted
position. There
are alternative
methods for
preparing a
streak.

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References
Basic

Practical Microbiology 2006


Society for General Microbiology ISBN 0
95368 383 4

Google

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resources from images

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Program

Created by Dr.T.V.Rao MD for the


benefit of Medical Microbiologists for
learning basic principles in inoculating the
culture plates, as a Mission for Universal
education of skills in Microbiology

Email

doctortvrao@gmail.com
5/7/16

Dr.T.V.Rao MD

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