1 ABSTRACT

The objective for this experiment is to know the morphology of the bacteria that
contain on the water. First of all, the sample of waters was taken from three
different locations. The locations of the sample waters that was taken for this
experiment was from the lake, the pond and the drain. Each of the sample need
to be cultured and undergo staining process before it can be observed under the
light microscope. The technique used is spread method under the pure culture
technique. All of the samples must be spread under laminar flow. After spreading,
the samples must be incubated for at least 24 hours inside an incubator. The
sample then will undergo staining process in order to identify the morphology of
the bacteria that contains on the sample. The samples from the lake and pond
shows that the bacteria presents are in rod shape while the bacteria from the
drain are in coccus shape.

2 INTRODUCTIONS
The experiment is conducted to observe the morphology of the microorganism
that are present in our samples. There are four samples that we are used in this
experiment which are samples from lake, pond, and drain water. Pure culture
technique was applied in this experiment to culture the sample. The pure culture
technique that we used is spread plate method for lake, pond, and drain water.
The samples are observed under light microscope after 24 hours incubated to
identify the shape and gram reaction of the microorganism present. Staining
method was applied on each sample to make it more clearly to observe under
the microscope.
Lake and pond is a small bodies of water that surrounded by land. There are
many types of microscopic life that can be found in a lake and pond. Microscopic
life is a tiny creature that cannot be seen with naked eyes. It only can be observe
by using a microscope. Organisms that found in the lake give balancing in the
ecosystem. Ecosystem is a place for biological community occurs in some locale
which the physical and chemical factor that make up its abiotic environment.
Drain is something that is used for removing a liquid from a place or container.
There is also has many types of bacteria that we can found on the drain water.
Not only the gram negative bacteria but also gram negative bacteria because
the water that flow comes from many types of sources.
So, it shows that anywhere and everywhere, the bacteria can be present.
3 OBJECTIVES
1

To identify the typical microorganisms found in the pond and food i.e.
protozoan, algae, and cyanobacteria.

To prepare a stock culture of an organism using isolates from mixed cultures

prepared on an agar streak plate and/or spread plate.

To observe the morphology of the bacteria present in the sample.

4 THEORY

There are many microorganism that we can see when we observe fresh water
sample under optical microscope. We can obtain water samples from ponds,
lakes, rivers, aquarium tanks or even from a small water puddle.
One of the microorganism that we can observe is bacteria which belongs to the
Kingdom Monera. Next, there are also microscopic animals called rotifers which
belong to the Kingdom Animalia. Protozoans and Algae are also present in
freshwater. Both belong to the Kingdom Protista (Microbus, 2015).
The smallest microorganisms possible to observe under optical microscope are
bacteria. Essentially, they exist in any kind of water specimen. However, their
quantities are much larger in waters that contain a high amount of decaying
organic substances. Bacteria are unicellular. They are classified as prokaryotes
due to the lack of nuclear membrane (Carboni, 2006).
One of the bacteria commonly found in fresh water is the Blue-Green Algae or
Cyanophyceae. They are amongst the earliest organisms to exist on Earth and
they are able to produce their own food using photosynthesis. Most bacteria are
small and clear. For blue-green alga, they are large and appear segmented.
These algae are often found in little groups consist of 2-4 cells or more. They can
also easily found in a mixture with filamentous algae like the spirogyra. Like
other algae, blue-green algae are formed of many individual cells. This means,
they live in a colony. Hence, they are not considered to be a single multicellular
organism. Since they have unique characteristics, sometimes they are called
eubacteria (Carboni, 2006).
Protozoans are categorized depending on their locomotion. There are four types
of protozoa. The first is the phyla Mastigophora. They move using a long
organelles that looks like a tail called flagella. Next is the Ciliophora. From their
name, it not surprising they have hundreds of cilia which beat in harmony. The
cilia act like little oars to move them through the water or liquids. The Sarcodina
includes the simple looking Amoebas. Their movement is similar to a flowing
globule of jelly using a protruding "pseudopod", or false foot. The last type of
protozoan is the Sporazoans. They are very small spore-like that doesnt seem to
have any kind of specialised movements. They can be pathogenic like those that
cause malaria disease. According to the scientists, there are approximately more
than 50,000 different species of protozoans (Microbus, 2015). However, there
are many new protists waiting to be found as new species are identified regularly
(Microbus, 2015).
Finally, there are microscopic animals or critters. They reside in the Kingdom
Animalia because they are multicellular animals. Some, like the rotifers appears
similar to common protozoans and even have cilia but they are made of many
cells and have organs like other animals (Microbus, 2015). In terms of size,
rotifers are slightly larger than the average protist. Rotifers can be found from
the observation of fresh water samples such as pond water under microscope.
They feed on bacteria, small protozoa and unicellular algae. Rotifers can move or
attached themselves on a surface using a foot which appears like a branched tail
(Carboni, 2006).

5 PROCEDURES

Spreading Method
1

A sample of water from the lake is collected.

Under the fume hood, both of the hands are sterilized by spraying alcohol.

01ml of the lakes water are pipetted and is poured onto the middle of the
agar surface.

An L shaped stick is placed on the agar surface and used to push and the
liquid is spread all over the surface.

The L shaped stick is lifted away after finished spreading for 5 to 10 seconds
and the cover is covered on the petri dish.

The petri dish is closed and taped with labeled as Sample A, later it is
incubated in an inverted position.

These steps are repeated by using different samples of water such as water
from the pond as Sample B and water from the drain as Sample C.

Staining Technique
1 The glass slide is sterilized by passing it through a burning flame.
2 The surface of sample A is scraped gently by using a cotton swab.
3 After that, sample A from the wooden cotton bud is then transferred on the
sterile glass slide.
4 A few drops of crystal violet are dropped onto the sample A and after 30
seconds, excess crystal violet is rinsed off with distilled water.
5 Then, a few drops of iodine are dropped onto the sample A and again after
waiting for 30 seconds, the excess iodine is rinsed off with distilled water.
6 Generous amount of alcohol are sprayed onto sample A and after waiting for
30 seconds rinsed it off with distilled water.
7 A few drops of safranine are dropped onto sample A and this time after
waiting for 1 minute, the excess safranine is rinsed with distilled water.
8 The sample A was let to dry for a few minutes, later cover slide was placed
onto the sample.
9 The glass slide is then placed on the light microscope to be observed.
10 All observation was later been recorded.
11 Step 1 until step 11 was then repeated with incubated sample B and Sample
C.

6 APPARATUS

Sterile cotton swab, bacterial samples from the various environmental

exposures, petri dishes with agar, parafilms, incubator, inoculation loop, glass
slides, Bunsen burner, Lugols solution, ethanol (decolorizing agent), safranine,
distilled water and Microscope(MT4200H,Japan)

7 RESULTS

Lake water

Pond water

Drain water

Lactobacillus sp.

Lactobacillus sp.

Streptococcus sp.

Cell shape
morphology

Rod shape

Rod shape

Coccus

Cell arrangement
morphology

Diplobacillus

Diplobacillus

Streptococcus

Purple

Purple

Purple

Gram-positive
bacteria

Gram-positive
bacteria

Gram-positive
bacteria

Draw
representative
field

Microorganism

Cell color

Gram reaction

8 DISCUSSIONS

There is an abundance and variety of microscopic life found in pond water, drain
water and lake water. The experiment was conducted because to find the major
microorganisms that live in the sample and to find their gram reaction. These
organisms are essential to the balance of the pond ecosystem. Based on the
experiment we had done, we found that Lactobacillus is the major
microorganisms that live in the lake water and pond water whilst Staphylococcus
sp. are the major microorganisms that live in the drain water.
We had conducted gram staining experiment to find out type microorganisms
and their gram reaction. Observation has been made. For Lake and Pond water
samples, we found out that Lactobacillus under gram positive bacteria. The
purple in colour are the major colour that can be seen through microscope.
Crystal violet stained the bacteria cell walls. To form the crystal violet-iodine
complex, Iodine is subsequently added as a mordant, so that the dye cannot be
removed easily. This step is commonly referred to as fixing the dye. However,
subsequent treatment with a decolourizer, which is a mixed solvent of ethanol
and acetone will dissolves the lipid layer. But it does not affect Lactobacillus
because this microorganisms have a thick wall of peptidoglycan and a small
amount of lipid bilayer. We observed that, this microorganisms is rod in shape.
Diplobacillus are the cell arrangement morphology for the Lactobacillus.
Next, for the drain water sample, we found out that Streptococcus sp. are the
major microorganisms that live in the sample. We also observed that this
microorganisms is gram positive bacteria the same like Lactobacillus. It has thick
of peptidoglycan and a thin layer of lipid bilayer. It has coccus shape and their
cell arrangement morphology are streptococcus.
This species grow up in a chain. This type of microorganisms can live in any
condition and any place. It do not have any specific environment.

9 CONCLUSIONS
Based on the experiment that has been conducted, the main objective was
successfully accomplished and the result was matched with the theory that has
been stated. . The first objective of conducting this experiment is to identify the
typical microorganisms found in the pond, lake and drain. By doing this
experiment, we are able to identify the type and name of the microorganisms
found in our three samples which are lake water, pond water and drain water.
The second objective is to classify the microorganism found according to the
taxonomy. Spread plate method was used to get single isolated pure colonies.
Aseptic technique was applied to avoid contamination of sterile media and
equipment during cell culture. The presence of bacteria can be observed with
their morphology and gram staining reaction.
By applying a gram staining method, we can determine the bacteria group either
it is gram positive or gram negative. Gram positive bacteria will remain as purple
colour and does not easily decolorized by alcohol. Gram negative bacteria will
change to reddish or pink colour because the purple colour of crystal violet
decolorized then counterstained with a safranin solution. Based on the result, all
the bacteria that has been observed in the lake, pond water and drain water
shows gram positive bacteria. The morphology of bacteria in these three samples
has been observed under light microscope. The microorganism found in the lake

water and pond water is Lactobacillus sp. and it has rod shape. Next, the
microorganism found in the drain water is Sreptococcus sp. which has
streptococcus shape.

10 RECOMMENDATIONS
In this experiment, there are some precautions steps recommended in order to
improve the quality of the results. First, make sure the experiment is performed
in the laminar flow cabinet to avoid contamination. The petri dishes or plate will
easily contaminate when we bring the plates out from the laminar flow cabinet.
Second, make sure to sterilize all cultures at 121C using the autoclave before
disposal or cleaning of lab ware to avoid contamination hazard. Third, when
conducting an inoculating loop, make sure it does not overheat when touching
the specimen because it can destroy and kill the bacteria. Fourth, do not apply
too much pressure while doing spreading method of the bacterial on the nutrient
agar medium because this will lead to breakage of the agar medium. Next, make
sure to wear glove and spray some alcohol on the glove to ensure free from any
bacterial infection. Lastly, make sure clean the table and working area to prevent
from a bacteria still life because it can cause infection to us.
For future experiment, it is recommended to use guar gum to substitute with the
agar as the price is cheaper and affordable. Next we can also use pour plate
method instead of spread methods used in the experimental procedures.
Besides, it is advisable for the laboratory to use a small turntable to make the
spread plate process easier rather than using L-shaped bend rod.

11 REFERENCES
1

Microbus. (2015). Pond Water Critters That You Can See With a Microscope:
Protozoans and Small Animals. Retrieved from the Microbus website:
http://www.microscope-microscope.org/applications/pond-critters/pondcritters.htm

Carboni, G. (2006, December). Small Freshwater Organisms. Pianoro

(Bologna), Italy: Fun Science Gallery. Retrieved from the Fun Science Gallery
website: http://www.funsci.com/fun3_en/guide/guide1/micro1_en.htm