MEDIA PREPARATION, UBUQUITY & CULTURAL

CHARACTERISTICS AND ISOLATION OF BACTERIAL

CULTURES
REYES, KIM RAFAELLE E.
Mapua Institute of Technology
Intramuros, Manila
Philippines
kimayna@yahoo.com

ABSTRACT
These series of experiments are conducted to give us a wider perception to the nature of
microorganisms and to be familiarized with the common terminologies and techniques in the
field of Microbiology. To start off, we disinfected our working stations and sterilized our
apparatuses. We then prepared a culture medium which will supply nutrients to the bacteria. The
human mouth will be the source of bacteria for this experiment. When the culture medium is
ready, we started collecting bacteria from the mouth using a cotton swab. Upon inoculating the
bacteria to the petri dish, Aseptic technique is then applied to prevent contamination. For the
subculture, an inoculating loop is heated at an optimal temperature. The loop will then transfer
a colony to a slant test tube which will serve as our subculture.

INTRODUCTION
A characteristic of microorganisms is their ability to grow and form a population of
organisms. One of the results of microbial metabolism is an increase in the size of the cell. The
many requirements for successful growth include those both chemical and physical. In order to
grow successfully, microorganisms must have a supply of water as well as numerous other
substances including mineral elements, growth factors, and gas, such as oxygen. Culture medium
is then made in order to grow microorganisms inside the laboratory. The most commonly used
culture media is the Agar, a gel-forming polysaccharide (70% agarose and 30% Agaropectin)
which solidifies bacterial media.

Bacteria grow on solid media as colonies. A colony is defined as a visible mass of

microorganisms all originating from a single mother cell. Key features of these bacterial colonies
serve as an important criteria for their identification. Some criteria that are used for
characterization of bacterial growth are appearance of the colony surface, consistency/texture,
color of the colonies (pigmentation) and opacity of the bacterial colony.

Pure cultures are best obtained by using solid media, by streak plate or pour plate
method. Streak plate, if properly done, is the most practical method. In the streak plate method, a
loopful of the inoculum is placed near the periphery of the plate with agar medium and spread or
streaked on the upper portion of the plate with parallel overlapping strokes. The inoculum is
streaked over other portions of the plate so that isolated colonies could be observed in the last
streaked area.

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 Bacterial isolation can be done using a general medium, wherein various bacteria can
grow, and selective media that allows growth of specific genera. Examples of general media are
nutrient agar (NA), tryptic soy agar (TSA), and brain heart infusion agar (BHIA). Examples of
selective media are thiosulfate citrate bile sucrose agar (TCBS) for vibrios, and glutamate starch
phenol red agar (GSP) for aeromonads and peudomonads.

MATERIALS AND METHODS

Disinfection of workstations and sterilization of apparatuses. Before conducting any

experiment, the work station is disinfected using disinfecting spray (in this case Lysol is used).
Petri dishes and test tube which will be acting as a vessel to our bacterial colony are sterilized by
autoclaving. Test tubes are sealed with prepared cotton plugs and are foiled to avoid
contamination.

Preparation of media. For this experiment two growth media are prepared: the Nutrient Agar
and PDA (Potato Dextrose Agar). The amount of powdered medium needed in the preparation is
specified at the label. The required mass of agar is then calculated using ratio and proportions as
per specified by the manufacturer. The agar is melted for about 15 minutes with continuous
stirring.

Pouring of agar into sterilized petri dish and slant. After so, the agar is poured onto the petri
dish and test tube (for slants) and then cooled. They are incubated at room temperature for 24
hours before checking for contamination.

Inoculation of bacteria. The bacteria used in the experiment is acquired from the mouth using a
cotton swab. The bacteria is then inoculated into the petri dish by streak plate method. The petri
dish is then refrigerated.

Characterization of bacterial colonies. The colonies are characterized by form, margin and
elevation. (Figure 1)

Isolation/Subculture of bacterial colony. A colony in the mother culture is selected among all
the other colonies (the colony subjected to isolation should be small to fit the inoculating loop).
The loop is heated via alcohol lamp (you will know if the heat is just fine when you are able to
withstand the heat when tested to your skin). Be careful because bacteria may die in high
temperature. The selected colony is touched by the loop and is spread at the slants.

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RESULTS AND DISCUSSION

(1) (2)

1: Microbial growth on Nutrient agar

2: A closer look on the bacterial colonies

(3) (4)

(3) & (4): Slant (Subculture)

Characterization of Bacterial Colonies

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CONCLUSIONS
This study demonstrates that

ACKNOWLEDGEMENT
This research was supported by

REFERENCES
[1] Speit, R.J., C.D. Cornell and U.T. Kaplan, Renewable Energy Conversion and Utilization in
ASEAN Countries, International Journal of Science and Technology, Vol. 23, No. 2, 2000,
pp. 125-134.
[2] Cornell, C.D. and U.T. Kaplan, Sources of Energy, Elsevier, 2003, pp 125-134.