ISOLATION AND CULTURE OF PROTOZOA

Klarys Abainza, Jessica Del Mundo, Emmylou Nicolas, May Orozco, and Sophia Sesbreo
BS Biology 3A. Bicol University, Department of Biology

ABSTRACT
The use of cell culture will help determine the behaviour, metabolic activity, regulation, gene expression and other
features of a particular cell. Protozoans were the study organisms because of their ubiquitous distribution, high
reproductive rate, ease of culturing and accessibility of experimental manipulation. This activity was conducted to
understand the conditions that limit cells' ability to survive and proliferate in vitro and learn the simple techniques in
cell culture. Two hay infusion medium were prepared for this activity, one media was added with pond water and
the other was sterilized and was used for obtaining a pure culture. After a week, the hay infusion media with the
pond water was observed under a light microscope and selection of a protozoan to be isolated was done, then the
glass slide with the isolated protozoan was washed into glass bottles containing the sterilized hay infusion media.
The protozoan isolated was fed with E. coli and was monitored once a week. The isolation procedure was done until
a pure culture was obtained. Proper handling and use of aseptic techniques should be observed to gain the expected
pure culture that is being aimed. Less talk less contamination; possible contamination factors should be minimized.
Isolating pure cultures will allow us to study, understand and give focus on the mechanisms of a specific organism.

Keywords: isolation, cell culture, pure culture, protozoa

INTRODUCTION
Cell isolation and culture are essential tools for the life sciences today specifically in the study of cell
function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of
resolution under the microscope. Although the organelles and large molecules in a cell can be visualized
with microscopes, understanding how these components function requires a detailed biochemical
analysis. Most biochemical procedures require obtaining large numbers of cells and then physically
disrupting them to isolate their components. If the sample is a piece of tissue, composed of different types
of cells, heterogeneous cell populations will be mixed together. To obtain as much information as
possible about an individual cell type, biologists have developed ways of dissociating cells from tissues
and separating the various types. These manipulations result in a relatively homogeneous population of
cells that can then be analyzedeither directly or after their number has been greatly increased by
allowing the cells to proliferate as a pure culture. In its simplest form, cell culture involves the dispersal
of cells in an artificial environment composed of nutrient solutions, a suitable surface to support the
growth of cells, and ideal conditions of temperature, humidity, and gaseous atmosphere. Using cell
culture, one can determine the behaviour, metabolic activity, regulation, gene expression and other
features of a particular cell (Alberts, 2002; Detera 2013).
Many animals and plant cells survive and proliferate in a culture dish if they are provided with a suitable
medium containing nutrients and specific protein growth factors (Alberts, 2002). Ciliates are used as test

organisms in laboratory experiments because of their ubiquitous distribution, high reproductive rate, ease
of culturing and accessibility of experimental manipulation. Paramecium are ciliate protozoan, the whole
body is covered with cilia, which help the organisms to swim forward, backward and turn. They are
shaped like prolate spheroids of 250mm length. It generally feed on bacteria, other small cells, yeast or
small alga. According to Guiffre, et. al. (2011), a sensory apparatus allows the paramecium in detecting
temperature, light, and a variety of attracting and repelling chemical substances. Their one complex cell, a
eukaryote, conducts all of the organism's basic functions. It doesn't divide work between different tissues
or cells like an animal. Instead, each Paramecium is capable of an aerobic exchange, similar to breathing,
reproducing asexually by cell division, ingesting nutrients, and expelling waste. They grow rapidly and
naturally grow in freshwater environment, an environment that can be easily copy in the laboratory
(Detera 2013). This activity was conducted to (1) understand the conditions that limit cells' ability to
survive and proliferate in vitro and (2) learn the simple techniques in cell culture.

METHODOLOGY
Preparation of hay infusion media
1 L of tap water was boiled in a beaker and a handful of hay was added as the water comes to a boil. After
ten minutes, the beaker was removed from heat and was transferred in glass bottles and left at room
temperature for a week, and then 25 mL of pond water was added to the hay infusion media.
A sterilized hay infusion media was prepared as well which will be needed in obtaining a pure culture in
the later part of this activity. To prepare the sterilized hay infusion media, 1 L of tap water was again
boiled in a beaker and a handful of hay was added eventually. However, the amount of water and hay can
be increased. After ten minutes, the beaker was removed from heat and was placed in Erlenmeyer Flasks.
The prepared media was placed in an autoclave to ensure that any unwanted organism be killed to prevent
contamination. The media was again cooled down and was stored in a refrigerator until it is ready for use.
Isolation of protozoa
For the isolation of protozoa, a drop of water from the hay infusion media added with pond water was
placed in a clean glass slide. It was observed in a microscope and cotton fibers were placed in order to
slow down the movement of the protozoa. A micropipettor was then used to transfer a selected protozoan
to another glass slide. Since Paramecium was the most abundant, it was then selected for cell culture. As
long as it is guaranteed that the drop contains a pure culture, the slide was washed into a sterilized glass
bottle containing sterilized hay infusion media using a micropipettor. This was done inside the hood so as
to prevent contamination and to maintain the integrity of the pure culture. After washing, the isolated
protozoa were fed with Escherichia coli. The glass bottle was then placed in a clean and lighted area in a
room. The culture was checked once a week until a pure culture of the protozoa is obtained.

RESULTS AND DISCUSSION

It took us two trials to have a pure culture. Figure 1 shows the paramecium obtained in the pure culture
under an electric light microscope in a magnification of 400x. It generally fed on bacteria, other small
cells, yeast or small alga in this activity, E.coli was used. They were able to proliferate asexually and
have the size which is large enough to view their internal compartmentalization. There were no other
organisms observed but them. We were able to isolate the study organism, paramecium.

Paramecium under 400x magnification

CONCLUSION
Right handling and use of aseptic techniques should be observed to gain the expected pure culture that is
being aimed. Less talk less contamination; possible contamination factors should be minimized. Isolating
pure cultures will allow us to study, understand and give focus in a specific organism. Tissues can be
dissociated into their component cells, from which individual cell types can be purified and used for
biochemical analysis or for the establishment of cell cultures.

REFERENCES
Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland
Science; 2002. Isolating Cells and Growing Them in Culture. Available from:
http://www.ncbi.nlm.nih.gov/books/NBK26851/.
Detera, Marla. Cell Culture and Isolation of Paramecium using Hay Infusion Soup and Skimmed Milk.
StudyMode.com. Retrieved 10, 2013, from http://www.studymode.com/essays/Cell-Culture-AndIsolation-Of-Paramecium-39532300.html.