Gebze Technical University

Microbiology Laboratory
Report - 1

Ebru AKHARMAN 142204026

 16.03.2018

Laboratory Safety Rules, Sterilization and Media

Preparation For Bacterial Culture
AIM:

Sterilization is the killing or removal of all microorganisms, including bacterial spores

which are highly resistant. Sterilization is an absolute term, the article must be sterile
meaning the absence of all microorganisms. A healthy experiment in the microbiology
laboratory depends on the sterilization of the environment. The work area should also be
disinfected regularly. One of the most basic steps for microbiology is to prepare the
nutrient. Every organism must find in its environment all of the substances required for
energy generation and cellular biosynthesis. The chemicals and elements of this
environment that are utilized for bacterial growth are referred to as nutrients or
nutritional requirements. In the laboratory, bacteria are grown in culture media which
are designed to provide all the essential nutrients in solution for bacterial growth. The
aim of experiment is to learn sterilization and disinfection and prepare the nutrient.

INTRODUCTION:
Sterilization is a process of destruction of all forms of
living microorganisms from a substance.
1) While the majority of microorganisms are not
pathogenic to humans and have never been shown
to cause illness, under unusual circumstances a few
microorganisms that are not normally pathogenic
can act as pathogens. Treat all microorganisms—
especially unknown cultures—as if they were
pathogenic. A student who has a compromised
immune system or has had a recent extended illness
should talk with the instructor before working in the
microbiology laboratory.
2) All materials, media, tubes, plates, loops, needles,
pipetes, and other items used for culturing
microorganisms should be sterilized by autoclaving.
Otherwise, use commercially sterilized products.
Understand the operation and safe use of all
equipment and materials needed for the laboratory.
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3) Use a disinfectant, such as a 10% bleach or 70%
ethanol solution, to wipe down benches and work
areas both before and after working with cultures.
Also be aware of the possible dangers of the
disinfectant, as 70% ethanol can catch fire around
open flame or high heat sources. Bleach, if spilled,
can ruin your clothing. Either alcohol or bleach can
be dangerous if splashed in the eyes. Students
should know where the nearest eyewash station and
sink are located.
4) Use a disinfectant soap to wash your hands before
and after working with microorganisms.
Nondisinfectant soap will remove surface bacteria
and can be used if disinfectant soap is not available.
Gloves may be worn as extra protection.
5) Use pipette bulbs or pipetting devices for the
aspiration and dispensing of liquid cultures.
6) Never eat or drink in the laboratory while working
with microorganisms. Keep your fingers out of your

mouth, and wash your hands before and after the
laboratory activity. Cover any cuts on your hands
with a bandage. Gloves may be worn as extra
protection.
7) All cultures, chemicals, disinfectant, and media
should be clearly and securely labeled with their
names and dates. If they are hazardous, label them
with proper warning and hazardous information.
8) All items to be discarded after a class, such as
culture tubes, culture plates, swabs, toothpicks,
wipes, disposable transfer needles, and gloves,
should be placed in a biohazard autoclave bag and
autoclaved 30 to 40 minutes 121° C at 20 pounds of
pressure. If no autoclave is available and you are not
working with pathogens, the materials, can be
covered with a 10% bleach solution and allowed to
soak for at least 1 to 2 hours.
Disinfection is the destruction of pathogenic and
other kinds of microorganisms by physical or
chemical means. Disinfectants are chemical
substances used to destroy viruses and microbes
(germs), such as bacteria and fungi, as opposed to an
antiseptic which can prevent the growth and
reproduction of various microorganisms, but does
not destroy them. The ideal disinfectant would offer
complete sterilization, without harming other forms
of life, be inexpensive, and non-corrosive.
Unfortunately ideal disinfectants do not exist. Many
disinfectants are only able to partially sterilize. The
most resistant pathogens are bacteria spores but
some viruses and bacteria are also highly resistant to
many disinfectants.
Every organism must find in its environment all of
the substances required for energy generation
and cellular biosynthesis. The chemicals and
elements of this environment that are utilized for
bacterial growth are referred to as nutrients or
nutritional requirements. Many bacteria can be
grown the laboratory in culture media which are
designed to provide all the essential nutrients in
solution for bacterial growth.
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16.03.2018
Starch agar:
Starch agar is a general-purpose, nutrient
medium used for the cultivation of microbes.
Inclusion of starch makes it a rich medium for
those bacteria possessing the enzyme alpha-
amylase, which breaks starch down to its
component glucose molecules. This medium,
therefore, is useful for the detection of alpha-
amylase. Starch combines with iodine to form a
blue-brown color. If the microbe produces alpha-
amylase, the medium in the vicinity of the growth
remains amber, indicating that starch near the
growth was digested by the alpha-amylase
produced by the microbes. Starch agar consists of
heat-stable digestive products of proteins
(called peptones), as would be found in nutrient
agar. These provide amino acids, minerals, and
other nutrients used by a wide variety of bacteria
for growth. In addition, the medium contains
soluble starch.
Nutrient Gelatin Medium:
Nutrient Gelatin is used to determine gelatin
liquefaction by proteolytic microorganisms.
Gelatin was the first gelling agent used to solidify
liquid culture media, thus bacterial counts could
be determined and isolation of pure cultures
obtained. However, when using gelatin
incubation temperature of 20 degree C was
required and it was observed that many
organisms were capable of liquefying the gelatin.
Agar eventually replaced gelatin as a solidifying
agent. Nutrient gelatin is recommended for the
taxonomic differentiation of the
Enterobacteriaceae and nonfermenting gram-
negative bacteria. The rate of liquefaction is used
as a differential characteristic. This medium is
primarily useful when investigating the
liquefaction characteristics of pure cultures of
organisms having minimal to no fastidious
nutritional needs.
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MATERIAL AND METHODS: RESULTS:
Nutrient Gelatin Medium: Bacterians, viruses, and microscopes can be seen
 Pepton 5 gr/L with life is very important for our lives. These
 Maya özütü 3 gr/L creatures can be beneficial or harmful to us.
 Jelatin 120 gr/L There are many rules to be aware of when
 pH is set to 6.8. working with these creatures. In this experiment,
important operations of the microbiology
1) The above materials are dissolved in hot water in
laboratory were performed. Sterilization and
a beaker and then transferred to the solution
covered tube. disinfection are at the beginning of these
processes. If it does not work clean enough, this
may be bad for the person who is both to try the
Starch agar:
experiment. Microorganisms are destroyed by
 Pepton 5 gr/L
sterilization. Disinfection removes some of the
 Maya özütü 3 gr/L
microorganisms. Then the medium was prepared
 Çözünebilen Nişasta 2 gr/L
for use. This medium has various rich features.
 Agar 15 gr/L
Many live on this medium, especially molds.
 pH is set to 7.2.
Therefore, after the medium is prepared, it is
1) The above materials are dissolved in hot water in autoclaved. The autoclave disrupts the proteins
a beaker and then transferred to the solution of microorganisms and neutralizes them.
covered tube.

2) After preparing the other materials to be sterilized

all the materials with the media are sterilized by
autoclaving for 15 minutes at 121 ° C. After
sterilization, the starch agar medium is expected to
cool to 50 - 60 ° C ve and the feed is poured into petri
dishes under aseptic conditions.

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