1.
STREAKING
CULTURE PLATES
IN BACTERIOLOGY
Dr.T.V.Rao MD
5/7/2016 Dr.T.V.Rao MD 1

2.
Think Before Inoculation
• Before inoculation,
important information is
written on the bottom of
the plates, close to the rim
• 1date of inoculation
• 2.temperature of incubation
• 3.duration of incubation
• 4.microorganism inoculated
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3.
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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4.
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 bacterium each.
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5.
The essential step in inoculating
culture plates
•There are several
essential precautions that
must be taken during
inoculation procedures to
control the opportunities
for the contamination of
cultures, people or the
environment
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6.
Prompt action with Optimal
utility
•Operations must
not be started until
all requirements
are within
immediate reach
and must be
completed as
quickly as possible5/7/2016 Dr.T.V.Rao MD 6

7.
Expose the inoculum in test tubes
and containers for minimalTime
• 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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8.
The Neck ofTest tube containing
inoculum to be heated briefly
•On being opened, the
neck of a test tube or
bottle must be
immediately warmed by
flaming so that any air
movement is outwards
and the vessel held as
near as possible to the
horizontal
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9.
Exposure to Environment should
limited to minimum
•During manipulations
involving a Petri dish,
exposure of the sterile
inner surfaces to
contamination from
the air must be limited
to the absolute
minimum5/7/2016 Dr.T.V.Rao MD 9

10.
Work with absolute sterility
• The 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 non-sterile
surfaces, e.g. clothing, the
surface of the working area,
the outside of test
tubes/bottles
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11.
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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12.
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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13.
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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14.
Perfect the art handling the
Inoculating loop
• Draw the rest of the
wire upwards slowly up
into the hottest region
of the flame,
(immediately above
the light blue cone). .
Hold there until it is
red hot.5/7/2016 Dr.T.V.Rao MD 14

15.
Heat and cool the Loop
• Ensure the full
length of the wire
receives
adequate
heating. .
Allow to cool
then use
immediately.
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16.
Handle the Inoculating loop
Do not put the
loop down or wave
it around..
Re-sterilise the
loop immediately
after use.
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17.
Be careful some times the Loop may not
contain inoculum must redraw the inoculum
• If a loop does not hold 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 puncturing the
skin.
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18.
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
Petri dish in such a way that
colonies grow well
separated from each other.5/7/2016 Dr.T.V.Rao MD 18

19.
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20.
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. .
Flame the loop and
allow to cool.
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21.
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22.
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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23.
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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24.
Do not Practice the
way
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25.
Practice for
Perfection• Replace the 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 solid
medium.
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26.
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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27.
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.
Turn the dish through
90° anticlockwise.
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28.
Streak the Plates in a defined
Manner
• With the cooled loop
streak the plate from
area ‘ across the
surface of the agar in
three or four parallel
lines . Make sure that
a small amount of
culture is carried over.
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29.
Streak the Plates in a defined
Manner
• 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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30.
Carry with further inoculations
•Remove the loop and close
the Petri dish. . Flame the
loop and allow to cool.Turn
the dish through
90°anticlockwise and streak
loop across the surface of the
agar from ‘ into the centre of
the plate
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31.
Complete the work Close the petri dish
• Remove the loop and
close the Petri 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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32.
References
•Basic Practical Microbiology© 2006 Society for
General Microbiology ISBN 0 95368 383 4
•Google resources from images
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33.
•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
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