BIOLOGY 3

MICROBIAL DIVERSITY
LABORATORY REPORT NO. 8
Hands-On Protistology Part II: The Ciliates, Flagellates, Amoebae, and possibly Algae
prepared by EMERY MYLES B. FLORES

A. Method Summary (in your own words and in paragraph form)

I. Preparation of Hay Infusion

The class poured tap water and stuffed leaves from a variety of species into a
beaker. We then boiled the leaves on a hot plate at 345 C. After 6 hours of boiling, I
poured out some of the water in the beaker and removed most of the pond leaves. I
poured half a bottle of pond water into the beaker. The beaker was then left to set aside
on a rack with ambient lighting.

II. Microscopic Examination

I borrowed and washed a depression slide and a cover slip from the lab. I used a
dropper to put 2-3 drops of the liquid from the hay infusion into the depression slide. I
then used forceps and scissors to cut a small piece of the leaf on the slide. I added a
small strand of cotton and covered the slide with a cover slip. I observed the
microorganisms in the slide under a microscope. Using Sir Millard’s provided guide, I
attempted to identify the microorganisms. I prepared multiple samples. I washed the
slides and coverslip with tap water after preparing each sample. I repeated this process
on two occasions: one with Sir Millard’s prepared hay infusion without leaves and the
other with the one the class prepared.

B. Observations / Results (photographs with captions, text, paragraphs, etc.)

I. Preparation of Hay Infusion

Figure 1.1 Boiling Hay Infusion

 Figure 1.1 shows the hay infusion boiling process on a hot plate at 345 C. We
waited 6 hours before replacing the tap water with pond water.

Figure 1.2 Sir Millard’s Prepared Hay Infusion

Figure 1.2 shows Sir Millard’s prepared hay infusion. Sir Millard's prepared hay
infusion is a rich broth made from leaves and pond water. It was yellow and had a
pungent odor. While waiting for the class’s hay infusion to boil, we observed his hay
infusion under a microscope.

Figure 1.3 Pouring the pond water into the beaker.

Figure 1.3 shows me pouring half a bottle of pond water into the hay infusion.
The pond water was sourced from the local ponds in Argao. Most of the leaves have
been removed. After this step, we placed the hay infusion on a rack with ambient
lighting and waited 2-4 days before examining it under a light compound microscope.
 II. Microscopic Examination

Figure 2.1 shows a depression slide I prepared for microscopic examination

using a compound microscope. It contains a small amount of cotton fibers and a few
drops from the hay infusion.

Figure 2.1 A depression slide containing drops from the hay infusion and cotton fibers.

Shown in Figure 2.2 are pictures of the different types of protists I observed
under the microscope. In Sir Millard’s hay infusion, I mostly observed ciliates like
Paramecium and flagellates such as Euglena. The Paramecium moved around the slide
quickly but the cotton fibers slowed them down. Some Euglena swam slower than the
Paramecium. I initially mistook a round Euglena as a ciliate because it moved rather
differently from the rest of the Euglena as if it was a leaf drifting in the wind. It also had a
similar shape to the Parameciums. However, after closer inspection, I discovered its
flagella and concluded that it was actually a Euglena.

Figure 2.2 Protists observed under the microscope.

 The 4 day-old hay infusion prepared by the class revealed ciliates, flagellates,
and amoeba. The hay infusion’s pungent odor was stronger now. The Parameciums
were now bigger and appeared to be “munching” various debris around the slide. The
Euglena were now stationary but still retained their vibrant green color from their
chloroplasts. My classmate, Paula Elumba, observed an amoeba in her slide. I took
pictures of it (Figure 2.2). It had an irregular shape and moved around like a blob of
slime. A ciliate kept touching it and it seemed as if the amoeba was trying to get away
from the Paramecium.

C. Discussion (explain what you saw / explain your results)

I. Preparation of Hay Infusion

Hay infusions are often used in biology labs to culture microorganisms from the
environment. The hay infusion is prepared by boiling hay in water, allowing the mixture
to cool, and filtering out the solid hay particles. After further research, I discovered that
boiling is done primarily to sterilize the mixture and facilitate the growth of
microorganisms for observation and experimentation. Boiling eliminates unwanted
microorganisms with high temperatures while inactivating harmful microorganisms that
may out-compete the organisms of interest (Tortora, Funke, & Case, 2018). It also
releases nutrients (Pelczar, Chan, & Krieg, 1986) and provides a consistent medium for
study (Prescott, Harley, & Klein, 2002). The resulting liquid is a nutrient-rich broth that
can support the growth of a diverse range of microorganisms. Hay infusions had a
pungent odor primarily because of the microbial activity that occurs during the
decomposition process. As the hay infusion continues to ferment and decompose over
time, the concentrations of these byproducts increase, leading to the characteristic
pungent odor. The specific odor depends on the types of microorganisms present and
the compounds they produce.

II. Microscopic Examination

When observing the hay infusion under a microscope, I expected to see a variety
of microorganisms, including bacteria, protozoa, and possibly even fungi. Bacteria are
usually the most abundant microorganisms in a hay infusion, and they can be easily
identified by their characteristic shapes, such as cocci (spherical), bacilli (rod-shaped),
and spirilla (spiral-shaped). Protozoa are single-celled eukaryotic organisms that can
move using flagella, cilia, or pseudopodia. They are larger and more complex than
bacteria and can be easily identified by their distinctive shapes and patterns of
movement. Fungi, on the other hand, are multicellular eukaryotic organisms that
typically grow as filaments called hyphae. I did not observe fungi in our sessions
because it can be difficult to identify them under a microscope without staining or other
specialized techniques.
 Paramecium ciliates are heterotrophs, which means they feed on other
organisms for their nutrients. They primarily consume bacteria and smaller protozoa
found in the hay infusion, using their cilia to create water currents that bring food
particles toward their mouth or oral groove. The ciliates I observed were mostly
Paramecium. They swam around the slide munching on debris that may have been
bacteria. They ranged from small round-shaped organisms to elongated oval-shaped
organisms.

Euglena is a genus of single-celled, eukaryotic organisms belonging to

Euglenozoa. They can display mixotrophic nutrition, which means they can obtain
nutrients through photosynthesis and heterotrophic feeding. Their green color comes
from their chloroplasts, which enable them to perform photosynthesis when light is
available, producing their energy and nutrients from sunlight and carbon dioxide.

Amoebas have a unique shape-shifting ability, which is enabled by their flexible

cell membrane and a gel-like cytoplasm. They do not have a fixed shape, and their
appearance constantly changes as they move and feed. Amoebas exhibit a
characteristic type called "amoeboid movement," where they extend pseudopods
(temporary, arm-like projections) to propel themselves and engulf their prey.

The amoeba we observed conflicted with a ciliate. Although ciliates can

occasionally prey on amoebas, it is not their primary food source. However, it may have
been the other way around. Some amoebas eat ciliates. Amoebas are opportunistic
feeders and consume a variety of food sources, including bacteria, algae, and other
protozoans like ciliates. This predator-prey interaction between amoebas and ciliates is
part of the intricate food web in aquatic ecosystems. Different microorganisms play
essential roles in maintaining the balance and cycling of nutrients.

REFERENCES

Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2018).
Brock Biology of Microorganisms (15th ed.). Pearson.

Pelczar, M. J., Chan, E. C., & Krieg, N. R. (1986). Microbiology: Concepts and
Applications. McGraw-Hill.

Prescott, L. M., Harley, J. P., & Klein, D. A. (2002). Microbiology (5th ed.). McGraw-Hill.

Tortora, G. J., Funke, B. R., & Case, C. L. (2018). Microbiology: An Introduction (13th
ed.). Pearson