Nutrient Agar Powder Preparation, Equipment Use and Aseptic Technique

I. Introduction

Culturing microorganisms is made possible by an agar plate, a Petri dish containing agar a
solid growth medium with addition of nutrients. Nutrient agar is a general purpose medium
which supports growth of a wide range of those non fastidious organisms. Microbiologists
basically been in the role of isolating a microbe from a mixed culture, then let isolated grow in a
pure culture, and lastly identify the isolate. Cultures may be grown in different kinds of media. In
which a medium is the material containing essential resources for growth which may be solid
with nutrients suspended in a liquid broth or it may be in a solidifying agent usually agar. An
agent extracted from a red seaweed is what we called agar. Specifically the nutrient agar is used
for microbial culturing or food medium. It contains different percentage volume or mass of
substances such as beef extract which provides carbohydrates, nitrogen, vitamins, salts; peptone,
helps control pH; agar, a carbohydrate used to solidify agent, distilled water, a reagent for
distributing food materials to growing colonies of microorganisms. With a favorable
environment will state the success of the growth of microorganisms given with adequate supply.

Science laboratory equipment has a huge importance of data gathering which will allow a
direct interaction to the students, and this aseptic technique isN a mandatory process employed
when handling bacterial cultures. According to Stevenson & Bykowski (2008), common
laboratory procedures that can reduce the risk of culture contamination (sepsis) is the aseptic
technique. Furthermore, Cappuccino & Welsch (2018), stated that sterility is the hallmark of
successful work in the microbiology laboratory.

II. Materials

The materials used in this activity were agar powder, sterile Petri dish, glass stir rod and all
and all
III. Methods

Before anything cleanliness was rest assured by the students guided with the aseptic
techniques. A mandatory to learn the perfect skills of inoculating bacterial specimens on agar
plates, in liquid broth, or in semisolid medium. Sterile inoculating needle was the instrument
used to transfer cultures and so aseptic technique was observed all the times. In the preparation
of the nutrient agar (NA) it was first measured the agar and distilled water into clean flask (23g
of agar added by 1000ml of distilled water resulted to 50 plates yields, 11.5g+500ml=25 plates,
then a 9.2g+400ml=20 plates and so with 4.6g+200ml=10 plates). Then a clean glass rod was
flame sterilized to stir the medium as it melted. Wearing a hand protection the beaker was put to
flame and was stir constantly, for 1 minute heating and was removed afterwards. With the used
of lab thermometer the temperature was monitored to 45-50 °C, then the melted agar was poured
to each sterile petri dish about 1/8-1/4 inch deep, and it was allowed to cool and set like stiff
gelatin at a room temperature for it to be ready for storage and use after the 15-20 minutes
sterilization.

For media sterilization, the students put at least 2 inches of water in the bottom of the
cooker then the baskets with media was put with wire racks. The knobs was tighten slightly until
all was securely fastened. Next the valve steam was opened in an upright position, all the air in
the pressure cooker was removed, and afterwards the valve steam was closed by turning it down.
The heat was regulated to keep the desired pressure. The media was removed from the cooker
and store for future use. The agar was stacked in an upside down in the refrigerator.

IV. Results

Preparation of
nutrient agar powder media
 Working safely in the laboratory requires proper use of laboratory equipment. Maintenance
accompanied with regular inspection can make a good outcome possible, for some instances the
hindrances are due to improper usages of specific equipment for such activity. Understanding on
how to make media, sterilize or how to prepare in general in which it requires the knowledge on
the following equipment; media, autoclave, Bunsen burner, micro incinerator, culture tubes, petri
dishes, wire loops and needles, pipettes, water baths, incubator, and refrigerator. Under media it
has three types, first is the broth wherein it is used to provide a growth medium for bacteria by
giving constantly and steadily amount of nutrients and it allows a bacteria to grow and quickly
reproduce. As well as there are semisolid and solid media, semisolid contains low media
concentration of agar and is used for motility determination of microorganisms, while solid
media is containing high concentration of agar and is used for identification and characterization
of microorganisms and its colonies. Medium is prepared by mixing nutrients and other materials
at a proper concentration, during preparation a solidification is also used and an example is an
agar but it does not show any nutritional value.

Autoclave is used in the preparation by putting foil on top of flask and placing on it, this is
used to carry out industrial and scientific processes which requires of temperature and pressure
elevation related to ambient pressure and/or temperature. The equipment was used after the
media containing agar is heated for the purpose of dissolving the agar. Most of the culture media
required 121° C for 20 minutes sterilization in an autoclave. The student observed that the
autoclave is one of the major tool used in preparing nutrient agar. Next tool is the Bunsen burner
along with wire loops and needles which also take part in the success of the experiment,
sterilization of wire loops for inoculation of agar slopes/deeps and small volumes of liquid media
as well as making streak plates. The water baths were used the suitable temperature for keeping
the melted agar media molten for use. The incubator was used for incubation of cultures.

Culture medium also known as culture medium can be in solid, liquid, or semisolid
designated to support the growth of population of microorganism. The substance that the
students used that lead the growth, support, and survival of microorganisms. Also it contained
the nutrients, factors that promote growth and any energy sources, buffer salts, minerals, metals,
and gelling agents in the solid type of media. Liquid culture media is called “broth’, while the
solid and semisolid is called the “agar”. For further division categories are divided as growth
media designated to grow the most heterotrophic microorganisms, transport media used in
preserved microorganisms, enrichment media, designed to increase the numbers of desired
microorganisms, and as well as the growth media. In this activity, agar was the culture media
used. Culture media must undergo quality control before it was being tested, in terms of physical
characteristics visual test for color, the visual test for clarity, the gel strength, pH of the finished
media and the checked for damaged. On the other hand, microbial characteristics was tested such
as the sterility to detect microbial contamination during the manufacturing process, temperature
and time selected for the sterility test incubation depends on the type of media. General purpose
media required 30-35°C for three days typically. There must be no growth demonstrated to pass
the sterility test. Culture media with microorganism was the challenge and the most important
test carried out in the microbiology laboratory. Suitability of the media was required for intended
use such as the demonstration of growth promotion of microorganisms. Type of culture was
carefully preserved within the culture of the laboratory.

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