EGE UNIVERSITY

GENERAL MICROBIOLOGY LABORATORY

(2023-2024 SPRING TERM)

ASEPTIC TECHNIQUE

Prof. Dr. Gülten GÜNDÜZ

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Media
A culture medium is a mixture of substances that promotes or support the growth of
microorganisms.
Culture media contain nutrients, energy sources, growth-promoting factors, minerals, metals,
buffer salts, and gelling agents (for solid media).

Based on consistency; Based on functional properties;

1) Solid media (1.5%-2% agar), 1) Basal media/Simple media
2) Liquid media 2) Selective media
3) Semi liquid media (0.5% agar) 3) Indicator/differential media
4) Enrichment media

A culture medium must be sterilised before use

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Liquid media Solid media

Agar Slant Agar Deep

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SLANT: Solid medium containing various nutrients and indicators. The slant gives the
microorganism a larger surface area on which to grow in a tube. Agar slants are also
useful for maintaining cultures, more so than stacks of Petri. Multiple cultures can easily
be placed in test tube racks and kept refrigerated. Microorganisms are inoculated onto a
slant using a loop or needle and grow on the surface of the agar.
DEEP: solid medium made up of various nutrients and indicators. This type of culture is
used for the growth of anaerobic bacteria, which grow in the absence of oxygen and are
inoculated by puncturing the medium with a needle.
BROTH: Liquid medium prepared with various nutrients and indicators. Allows the
growth of large quantities of microorganisms, the level of growth can be assessed by the
turbidity (cloudiness) of the culture. Microorganisms are inoculated into a broth using a
loop, needle or pipette.

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Steril petri Steril agar media Agar media in petri

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Culture
The growth of microorganisms in a media.

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The proper preparation:
1.No eating, drinking, or smoking in the laboratory.
2.Wear a lab coat to protect your clothing from contamination.
3.Always wash your hands before and after handling microorganisms.
4.Use disinfectants to clean the bench top before and after use.

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 ASEPTIC TECHNIQUE
Aseptic techniques are a set of
specialized procedures and routine
practices used in microbiology lab to
prevent contamination of samples
and cultures throughout the
analysis.

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Aseptic technique
It is essential for producing accurate and reliable results, as contamination can lead to false or
misleading results that could impact research findings and conclusions.

Aseptic technique is very important in microbiology to ensure safety and prevent cross-
contamination.

To create an aseptic environment, working areas must be disinfected, equipments must be

sterilized.

We must work close to the bunsen burner flame (no more than 10 cm).

In the lab, working beside a Bunsen burner creates an upward flow of air through convection,
lowering the risk that dust or other contaminants will settle on the sterile surface or equipment

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 The Bunsen burner
creates a radial sterile
field around it.

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! Specific Aseptic Techniques
-Wear a lab coat.
-Decontaminate your bench and hands with 70% alcohol.
-Organise your work area safely.
-Adjust your Bunsen burner correctly.
-Tie your hair back.
-Sterilise your inoculation equipment.
-Use sterile glass or disposable plastic pipettes and a pipettor to work
with liquids, and use each pipette only once to avoid cross
contamination.
-Do not unwrap sterile pipettes until they are to be used.
-Keep pipettes at the work area.
-Be careful not to talk, sing, or whistle while performing sterile
procedures.
-Perform experiments as rapidly as possible to minimize
contamination.
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Inoculation
Inoculation meaning in microbiology is that transfer from
culture for their growth.
In simple terms, inoculation is the process of introducing
microorganisms into a culture media, allowing them to
growth.
Inoculation can be done using equipments such as loops,
needles and pipettes.
-They must be sterilized before and after inoculation.
Generally, loop is used for inoculation to slant media, needle Pipet Loop and needle
is used for inoculation to deep media.

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Heat sterilisation
Hold the inoculating loop/needle in your
dominant hand like a pencil. To sterilize,
place it in the bunsen burner for at least 10
seconds. The entire wire must be heated
red hot. Use the center blue region of the
flame (not the top or bottom of the flame).

After the loop or needle is sterilized by

heating, cool in air, dip immediately into
ethanol (70%) and place it over the flame
again to evaporate the excess alcohol and
cool it on the tube wall for about 15
seconds before making any transfers.

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Inoculation;
❑With your non-dominate hand, pick up the parent tube by
grasping the tube just below the cap and lifting it out of the
rack.
❑Grasp the cap with the pinky and ring finger of your dominate
hand and gently twist the tube out of the cap keeping your
dominate hand still.
❑The cap is kept in your hand and never placed on the bench
top.
❑Heat the mouth of the open tube by passing it through the
flame of the Bunsen burner. Heating creates convection
currents, which carry airborne particles away from the mouth
of the tube, preventing contamination of the culture or
medium within.
❑Keep the tubes as nearly horizontal as possible. This prevents
unwanted microorganisms from entry.

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 Aseptic pipetting technique;
❑Light the bunsen burner.
❑without opening the sterile sleeve, look through the wrapper and check the volume of pipette.
Also make sure that the tip is not cracked or chipped and check the wrapper hasn't been damaged
in any way.
❑Open the wrapper and remove the pipette aseptically and insert the top, wide end into a pipette-
filler.
❑The cap of the tube containing the liquid culture is then opened with the right hand and the
mouth of the tube is passed through the flame.
❑Fill the pipette a bit above the capacity line desired and then slowly lower the meniscus to that
capacity line.
❑After the desired amount of liquid (inoculum) has been obtained by dipping the pipette into the
liquid, the mouth of the tube is again flamed.
❑The tube containing the sterile medium to be inoculated is taken in the left hand, the cap is
opened with the right hand and the mouth of the tube is flamed.
❑The inoculum is then pipetted out without being immersed in the liquid in the tube and the
mouth of the tube is flamed.
❑Dispose of the used pipette in an appropriate manner.

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 To the liquid medium

Pipette;

To the petri plate

Liquid medium

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Loop;
Liquid

Slant

Liquid media
Petri

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Loop;
Liquid

Slant

Petri
Petri

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Needle;

Petri

Culture petri Deep

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Incubator
An incubator is a device used to grow and maintain microbiological cultures.

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Incubation
Incubation is allowing microorganisms to grow under appropiate growth
conditions.

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https://www.youtube.com/watch?v=IiGGmHXLm1o&ab_channel=MCCCMicrobiology
https://www.youtube.com/watch?v=hdx48f7ORTM&ab_channel=MCCCMicrobiology

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Res. Assist. Ayşegül Kırmızıgül Peker
aysegul.kirmizigul.peker@ege.edu.tr

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