CULTURING ON PETRI

DISH
a practical approach to beginners of
Microbiology
Dr.T.V.Rao MD

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 What is a Petri Dish
• An agar plate is a Petri
dish that contains a
growth medium
(typically agar plus
nutrients) used to
culture
microorganisms
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 Purpose of Petri dish
• The petri dish was made for
separation. It was
developed for culturing
microorganisms while
separating them from
airborne contaminates., the
dish also assisted in
separating individuals from
disease.

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 History of Petri Dish
• In 1881, Fannie Hesse, who
was working as a technician
for her husband Walther
Hesse in the laboratory of
Robert Koch, suggested agar
as an effective setting agent,
since it had been
commonplace in jam making
for some time

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 Still Culturing is the Backbone of
Diagnostic Microbiology
• Bacterial culturing is the back bone of the
Microbiology laboratories in spite of many advances,
every graduate and post graduate and practising
Microbiologist should be proficient in the methods to
isolate identify the Microbes on tiny petri dish,
Individual microorganisms placed on the plate will
grow into individual colonies, each a clone genetically
identical to the individual ancestor organism
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THE ESSENTIAL STEPS IN INOCULATION OF
CULTURE PLATES

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 Steps in Inoculation
Step 1
• While wearing gloves,
sterilize an inoculating
loop by placing it at an
angle over a flame.
The loop should turn
orange before you
remove it from the
flame.
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Flaming Loop and Cooling for aseptic work

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 Step 2
•Remove the lid
from a culture
plate containing
the desired
microorganism
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 Step 3
•Cool the
inoculating loop
by stabbing it into
the agar in a spot
that does not
contain a
bacterial colony.
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 Step 4
•Pick a colony and
scrape off a little
of the bacteria
using the loop. Be
sure to close the
lid.
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 Step 5
•Using a new
agar plate, lift
the lid just
enough to
insert the loop.
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 Step 6
•Streak the loop
containing the
bacteria at the top
end of the agar plate
moving in a zig-zag
horizontal pattern
until 1/3 of the plate
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 Step 7
•Sterilize the loop again
in the flame and cool it
at the edge of the agar
away from the
bacteria in the plate
that you just streaked.

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 Step 8
• Rotate the plate about
60 degrees and spread
the bacteria from the
end of the first streak
into a second area
using the same
motion in step 6.
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 Step 9
• Sterilize the
loop again
using the
procedure in
step 7.
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Spread the Colonies with standard Protocols
•Rotate the plate
about 60 degrees
and spread the
bacteria from the
end of the second
streak into a new
area in the same
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Dr T.V.Rao MD 17
 Before you finish the planned work

•Sterilize
the loop
again
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 Place the Plates as Indicated in the
standard procedures
• Plates are incubated
upside down (agar up),
so that condensation
does not drip onto the
plate and interfere
with the developing
microbes.
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 Incubate Overnight as per the Standard
operative procedures
•Replace the lid and
invert the plate.
Incubate the plate
overnight at 37
degrees Celsius
(98.6 degrees
Fahrenheit).
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 You are successful if you observe pathogens on
streaks if Infected
•You should see
bacterial cells
growing in
streaks and in
isolated areas
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 Tips:
• 1. When sterilizing the inoculating loop, make sure
that the entire loop turns orange before using on the
agar plates.
• 2. When streaking the agar with the loop, be sure to
keep the loop horizontal and only streak the surface of
the agar.
• 3. If using sterile toothpicks, use a new toothpick
when performing each new streak. Throw all used
toothpicks away.
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 Streaking the Culture Plates
• The process involves spreading bacteria across
an agar plate and allowing them to incubate at a
certain temperature for a period of time.
Bacterial streaking can be used to identify and
isolate pure bacterial colonies from a mixed
population. Microbiologists use bacterial and
other microbial culture streaking methods to
identify microorganisms and to diagnose
infection.
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 Incubating and Viewing Plates
• Incubating the plates to promote growth of microbes
is an essential part of any microbiology investigation.
Incubating in aerobic conditions, and below human
body temperature, reduce the risk of encouraging
microorganisms (particularly bacteria) that could be
pathogenic to humans. Taping the lids on reduces the
chance that students will open plates when viewing,
but there are details below of how to kill plates
completely if this is still a significant risk.
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 References
• How To Streak a Bacterial Culture By Regina Bailey Biology Expert
About education

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• Program file crated by Dr.T.V.Rao MD for benefit
of Medical and Paramedical Professionals in the
Developing World
• Dr.T.V.Rao MD
• Freelance Clinical Microbiologist

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