Ed Christian D.

Monte
Exercise 1: Collecting and Culturing Protozoans
Zoo 111.1: Invertebrate Biology

ABSTRACT: Seven solutions were prepared from pond water and canal water as an inoculation source
for a Protozoa culture. Each solution had varying substrates and varying volumes of pondwater
medium. A five-day observation process was conducted. Using a microscope, each solution was
observed for certain protist taxa and population growth. Solution 1 yielded the greatest number of
protozoa per drop since it was the only solution not sterilized via boiling.

INTRODUCTION

Protists are a collection of unicellular eukaryotic organisms. According to multiple studies,

protists have been believed to be a common ancestor of multicellular plants, fungi, and animals.

(Pechenik, 2015). These organisms are microscopic in nature and are divided into informal subphyla.

These subphyla include the Protozoans; which are animal like in nature, plant-like Phototrophs, and

fungi-like slime mold. These terms, however, are not recognized as an official clade.

Protists can be found in a wide array of environments such as freshwater lakes, ponds,

puddles, and rivers. They can also exist in salty bodies of water such as Diatoms. These microorganisms

have also been found inside the gut of certain animals.

The term Protist can somewhat be misleading. It was German Scientist Ernst Haeckel that

suggested placing organisms that were not plants or animals into a new Kingdom. However, the

diversity of the Protist’s ultrastructure, life cycles, and evolutionary lineages with the help of molecular

data, was too much to contain into this Kingdom. In modern cladistic systems, the Kingdom Protista is

no longer recognized as a formal taxonomic category. The organisms inside this former clade was then

distributed among all kingdoms. The term, however, still serves as a general term to refer to these

unicellular microorganisms (Parfrey, 2010).

Protists are common in any kind of environment. The Philippines urban environment, being a

tropical country with a rainy season, can often be home to protists due to the widespread abundance

of puddles. These puddles if left for a long time can become an ideal thriving environment for these

microorganisms due to the warm 25˚C - 32˚C temperature (Atkinson, et al., 2003).
 In relation to this, the objective of the study was to create multiple cultures for these

microorganisms. Puddle water from multiple sources in Tacloban City was utilized as inoculation

sources and different substrates where placed in these cultures to determine the dynamics of protist

population.

MATERIALS AND METHODS

Sample Collection

Since tap water contained chlorine, it was not a viable option as the medium in which the

organisms are to be since chlorinated water is an ineffective growth medium for microorganisms.

(First et al., 2014). Instead, the researchers used pond water found in the Tickling garden inside the

University of the Philippines Visayas – Tacloban College Campus. The water found in this pond was

accumulated rainwater, which greatly decreases the chance of harmful chemicals being present in the

medium. Two litres of this rainwater were collected and was stored in an environment that was away

from direct sunlight. Figure 1 shows the pond from which the bulk of the growth medium was

collected.

A source for inoculation; rich in protists was also collected. The researchers utilized stagnant

canal water found along the road of Brgy. Del Pilar. Figure 2 below shows the actual canal water from

which the inoculation source was collected.

Figure 2: Inoculation Source, Puddle along the road of Brgy. M.H. Del Pilar
Preparation of the Solutions

Solution 1

Five hundred mL of pondwater was placed inside a 1.5 plastic bottle cut open in the middle.

A handful of dried grass was then placed inside the bottle.

Solution 2

Eight hundred mL of pondwater was put to a boil for 5 minutes. After 5 minutes of boiling, a

handful of dried grass was placed in a plastic bottle. The solution was placed inside a 1000mL beaker

since the plastic bottles was not enough to contain the entirety of the solution. After 2 days, it was

inoculated with the canal water.

Solution 3

One thousand mL of pond water was put to a boil for 5 minutes. Eight lightly crushed

uncooked corn kernels were then added while the solutions were still hot. The contents of the

beaker were then placed inside a plastic 1.5 bottle.

Solution 4

One hundred mL of pond water was put to a boil for 5 minutes. After boiling, 20 grains of

uncooked rice were then placed in the beaker. The contents of this beaker where then placed in a

plastic 1.5 plastic bottle.

Solution 5

Eight hundred mL of pondwater was put to a boil for 5 minutes. While the solution was still

hot, 2 pinches of boiled chicken egg yolk was added. The entirety of the solution was then placed

inside a cut 1.5 mL plastic bottle.

Solution 6

Two hundred mL of pond water was put to a boil for 5 minutes. Three pinches of skimmed

milk were added while the solution was still hot.

Solution 7

Two hundred-fifty ml of pond water was put to a boil for 5 minutes. Once the solution was

room temperature, 1 heaping tablespoon of dehydrated yeast was added.

Introduction of Inoculations

The solutions were left to settle and stabilize for two days. After this time period, inoculations

were then introduced to the seven solutions. Solution 2 was added 50mL, solution 3, 10 mL, solution

4; 10mL, solution five, six, and seven received 50 mL. For the five days of observation, 3 drops of Alaska

Evaporated milk were added to all the solutions on days 1, 2, 3, and 5.

Gauging Population Dynamics

The method that the researchers used for gauging the population dynamics was a process of

approximation in which the number of individual protozoans found under the LPO (100x

magnification) were counted. Spots where the protozoans seem to be most dense was the spots that

was used for this process. For 5 days, this process was conducted to all the solutions. The results for

this process was expressed as range values.

Results and Discussion

Population Dynamics

Using the process of counting protozoa under LPO (100x), the midrange values were taken

and plotted. Figure 3 shows the population Dynamics of the solutions during the 5-day observation.

Approximate Population of Protozoa Over Five Days

120

100

80

60

40

20

0
Day 1 Day 2 Day 3 Day 5

Solution 1 Solution 2 Solution 3 Solution 4

Solution 5 Solution 6 olution 7

Figure 3: Approximate Population of Protozoa Over Five Days

The solution that exhibited the most growth was solution 1. Solution 1’s medium source was

unboiled pond water. In this sense, bacteria are still present in the water. Protozoans are phagotrophs.

The rich bacterial environment gave way to a drastic increase to their population. (Sherr et al., 2002).

Solutions 2 to 7 medium was pondwater, however, was boiled, which killed the main food source of

the Protozoans. The bacteria that was present during microscopy sessions for solutions 2 to 7 were

introduced by the inoculation samples. The present bacteria population in these inoculations were

not enough to sustain the protozoans enough for reproduction, hence, the low population.
 Baker’s yeast and the egg yolk both contain proteins, supplemented with the addition of

evaporated milk, which is rich in lactose, could have been great substrates for bacterial growth.

(Buriling et al., 1917). However, due to their low population due to the boiling prior with to the

addition of the inoculation and the presence of phagocytic protists, the bacteria present did not

increase in population, which in turn, hindered the food source of the protists present.

Taxa Present in the Solutions

The most common protozoa found were the ciliated paramecium. They were present in all

the solutions. Figure 4 shows a Protozoa feeding on bacteria.

Figure 4: Paramecia under an Ocular Micrometer

Using an ocular micrometer, this organism’s size was 119 µm. Different species vary in size.

Generally, in nature, they are 50 – 330 µm (Dobell, 1932). In addition, a flagellated Euglena,

exhibited in Figure 5, and a Flagellated Phacus sp. Was also spotted.

 Figure 5: Euglena from Solution 1 (40x)

Figure 6: Phacus sp. From Solution 4 (40x)

 Heliozoans, protozoans with axopodia were also present but rarely spotted. Figure 6 shows 2

Heliozoans. These were found in solution 3 at day 3 of the observation.

Figure 6: Heliozoans (40x)

Conclusion

Protozoans, being phagocytic, need ample population of food such as bacteria to grow. The

addition of substrates rich in sugars and proteins can also aid their growth. Furthermore, in urban

freshwater environments such as puddles or canals, the taxa of ciliated paramecia are the most

abundant with respect to population.

To create a successful culture of protozoa and bacteria from a sterile/boiled medium, the ratio

between the inoculation source’s volume, medium, and food given should be closely monitored.

Furthermore, during microscopy sessions, it is best not to move the solution containers as to not

dislocate the protozoa concentration near the surface. In relation to this, samples to be placed on

glass slides should come near or on the water surface since it is at this space where protist and bacteria

concentration is greatest.
 APPENDIX

13/08/19 DAY 1
SOLUTION 1 Population: approx. 6-10 protozoa found in LPO
at any given time
Taxa found: Paramecium
Others: visible bacteria
SOLUTION 2 Population: 0-1 protozoans found in LPO at any
given time
Taxa found: Paramecium
Others: Bacteria are clumped in groups
SOLUTION 3 Population: 0-1 protozoans found in PLO at any
given time
Taxa found: Paramecium
Others: Bacteria present
SOLUTION 4 Population: 0-1 protozoans found in LPO at any
given time
Taxa: Paramecium
Others: Bacteria present
SOLUTION 5 Population: 0-1 protozoans found in LPO at any
given time
Taxa found: Paramecium
Others: Bacteria present.
SOLUTION 6 Population: 0-1 protozoans found in any given
time (LPO)
Taxa found: Paramecium
Others: Bacteria Present
SOLUTION 7 Population: 0-1 protozoans found at any given
time under LPO
Taxa found: Paramecium
Others: Bacteria present
 SOLUTION 1 Population: approx. 100 protozoa found in LPO
at any given time
Taxa found: paramecium
Others: visible bacteria
SOLUTION 2 Population: 0-1 protozoans found in LPO at any
given time
Taxa found: Paramecium
Others: Bacteria are clumped in groups
SOLUTION 3 Population: 0-1 protozoans found in LPO at any
given time
Taxa found: Paramecium
Others: Bacteria present
SOLUTION 4 Population: 0-1 protozoans found in LPO at any
given time
Taxa: Paramecium
Others: Bacteria present
SOLUTION 5 Population: 0-1 protozoans found in LPO at any
given time
Taxa found: Paramecium
Others: Bacteria present.
SOLUTION 6 Population: 0-1 protozoans found in any given
time (LPO)
Taxa found: Paramecium
Others: Bacteria Present
SOLUTION 7 Population: 0-1 protozoans found in any given
time under LPO
Taxa found: Paramecium
Others: Bacteria present
14/08/19 DAY 2
15/08/19 DAY 3
SOLUTION 1 Population: 100+ protozoa found at any given
time under LPO
Taxa found: Paramecium
Others: Ciliates dominate the culture
SOLUTION 2 Population: 2-3 protozoa found at any given
moment under LPO
Taxa found: Paramecium
Others: Bacteria found. Protozoa dominate
culture
SOLUTION 3 Population: 0-1 Protozoa found in LPO at any
given time.
Taxa Found: Paramecium
Others: Bacteria present
SOLUTION 4 Population: 0-1 Protozoa found under LPO at
any given moment
Taxa: Paramecium
Others: Bacteria present
SOLUTION 5 Population: 10-16 protozoa found at LPO at any
given time
Taxa found: Paramecium found
Others: Bacteria present
SOLUTION 6 Population: 0-1 Protozoa found at any given
time under microscope.
Taxa found: Paramecium
Others: Bacteria present
SOLUTION 7 Population: 5-8 Protozoa found at any given
time under microscope (LPO)
Taxa found: Paramecium
Others: bacteria present
16/08/10 DAY 4

SOLUTION 1 Population: approx. 100+ protozoans in

eyepiece in any given time (LPO)
Taxa found: Paramecium
Others: the population is very dense.
Paramecia dominate the solution in huge
numbers.
SOLUTION 2 Population: approximately 0-2 protozoans in
eyepiece at any given time (LPO)
Taxa found: Paramecium
Others: bacteria present. Presence of any
paramecium is scarce.
SOLUTION 3 Population: approx. 0-1 protozoans in eyepiece
at any given time (LPO)
Taxa found: Paramecium
Others: the sample does not exhibit any
protists. Bacteria present. Mostly bacillus.
SOLUTION 4 Population: approx. 0-1 protozoans in eyepiece
at any given time (LPO)
Taxa found: Paramecium
Others: bacteria present. Mostly are bacilli.
SOLUTION 5 Population: approx. 5-15 protozoans in
eyepiece at any given time (LPO)
Taxa found: Paramecium
Others: bacteria present.
SOLUTION 6 Population: 0-1 protozoans in eyepiece at any
given time (LPO)
Taxa found: Paramecium sp.
Others: Bacteria present
SOLUTION 7 Population: populous. Approx. 6-13 protozoans
in eyepiece in any given time (LPO).
Taxa found: Paramecium
Others: bacteria not that prevalent compared
to the other solutions
 SOLUTION 1 Population: approximately 30+ protozoa are
found in any given time (LPO).
Taxa found: Paramecium sp.
Others: The Paramecium found have become
much smaller in size.
SOLUTION 2 Population: approximately 0-1 are found at any
given moment.
Taxa found: Paramecium sp.
Others: bacteria are present.
SOLUTION 3 Population: approx. 2-3 protozoa are found in
any given amount of time (LPO)
Taxa: Paramecium sp.
Others: seems normal
SOLUTION 4 Population: approx. 3-10 protozoa are found in
any given amount of time (LPO)
Taxa: Paramecium sp.
Others: Protozoa size stayed relatively the
same.
SOLUTION 5 Population: 0-2 protozoa found in any given
amount of time (LPO)
Taxa found: Paramecium and
Heliozoa
Others:
SOLUTION 6 Population: 0-2 protozoa found in any given
moment (LPO)
Taxa found: Euglena, Paramecium
Others: Bacteria Present
SOLUTION 7 Population: 0-1 protozoan found at any given
moment (LPO)
Taxa found: Paramecium
Others: bacteria present
16/08/19 DAY 5
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