Republic of the Philippines

PARTIDO STATE UNIVERSITY

Camarines Sur
PS-2 BSSE 3A

Laboratory Report on the

Morphological Characteristics of the
Cultured Microorganism from the
Water Reservoir Sample

Submitted by:

MABANA, JICE RIZA

Submitted to:

ENGR. EMELINA R. PADAYAO

PS-2 Instructor
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A
I. Introduction

Microorganisms such as bacteria are ubiquitous and are noted to be not

visible to the naked eye. These omnipresent microorganisms live around us
and even inside of us. Bacteria are an essential part of the biosphere since they
help maintain the ecosystem’s balance, have a significant impact on health and
have several applications in different industries. Water reservoirs, either natural
or man-made, are vital sources of freshwater for human consumption,
irrigation, and industrial use. However, they often have and acquire diverse
microbial communities, including bacteria, fungi, and algae, which can impact
water quality and the environment.
Bacterial culture is a process of growing bacteria in or on a medium in a
lab environment (Meszaros, 2022). It is the basic method of microbiology, used
primarily for studying how a particular bacteria grows in certain methods and
conditions performed in a microbiology lab. By studying bacterial growth, we
can better understand how microorganisms interact with their surroundings,
adapt to different environments, and form colonies. Understanding bacteria’s
traits and biology is necessary in taking advantage of their benefits and
mitigating their risks. This knowledge is essential for designing effective
strategies for disease control, water treatment, and contamination prevention.
The study of these microorganisms involves culturing and characterizing
their morphological traits, which helps identify their roles in the environment
and potential risks to public health. This experiment is intended to isolate and
culture microorganisms from a water reservoir sample to observe their
morphological characteristics under controlled laboratory conditions. Using
selective media and incubation, we examined colony morphology,
pigmentation, and cellular structures.

II. Objective
The study seeks to:
• To familiarize students with proper laboratory techniques for culturing
and handling bacteria;
• To observe bacterial growth under different environmental conditions;
• To assess the number of colonies and their morphological traits on the
streaked agar plate; and
• To identify and describe the morphological characteristics (e.g., shape,
size, color, texture, arrangement, etc.) of the microbial colonies cultured
from the water sample

III. Methodology
A. Materials
• 1000 mL Collected Reservoir Water Sample
• 5 Test tubes
• 15 Prepared media
• 1 L-Shaped Cell Spreader
• 2 Micropipette
• 1 Alcohol lamp
• 1 Beaker
• Matchsticks
• Test tube rack
• Parafilm M
• Scissors
• Masking tape
• Incubator
• Biosafety Cabinet
• Single Tube Vortex Mixer
• PPE (Lab gown, Surgical gloves, Face mask)
• Alcohol
• Inoculating loop
• Marker
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A

B. Procedures
Week 1: Introduction to Bacteria Culturing and Media Preparation and
Demonstration of Laboratory Equipment
1. On November 8, 2024, students of BSSE 3A proceeded to the College of
Science, wherein they were taught by microbiologist Ms. Leizel Atole-Nieva
about the basics of bacteria culturing and media preparation.
2. After the hour-long lecture, the students were led inside the ParSU
Microbiology Testing Laboratory, and the microbiology equipment and
apparatus were introduced.
3. Ms. Leizel then divided the class into five (5) groups with each one tasked
to collect different samples.

Week 2: Hands-on Bacteria Culturing and Media Preparation

1. The second part of the experiment was conducted on November 21, 2024.
Seven (7) hours prior to the execution of the experiment, members of this
group collected 1000 mL of Reservoir Water Sample using a sterile
container to avoid contamination. The sample was taken from a well located
in Tigaon, Camarines Sur and it was obtained using the technique taught
by Ms. Nieva.
2. After wearing proper PPE, the group went inside the ParSU-MTL where the
necessary equipment was already prepared.
3. Before proceeding, Mr. Piano, one of the laboratory assistants, reminded
the students to observe proper aseptic technique by ensuring that the
culturing of bacteria must be done inside the Biosafety Cabinet and before
doing so, everything that will go inside the cabinet must be disinfected using
70% isopropyl alcohol. The exhaust filter of the stated apparatus was
situated at 101 Pa while the downflow filter was at 122 Pa.
4. Using a micropipette, 1 mL of reservoir water was taken from the sample
and diluted in one of the prepared test tubes. This was then taken outside
the cabinet and placed in the Single Tube Vortex Mixer for the substance to
be evenly mixed.
5. The test tube was then disinfected before being placed in the test tube rack
inside the Biosafety Cabinet.
6. This process was conducted four (4) more times to obtain the dilution
factors -1 to -5, and were labelled according to the stated factor with a
masking tape and marker.
7. Following the stated procedures, the group members were given ten (10)
agar plates with nutrient agar. An alcohol lamp and beaker with alcohol was
also prepared outside the cabinet with the L-rod placed in the beaker.
8. After disinfecting the plates, the members executed the spread plate
method. A sample of 0.1 mL was taken from the test tube with the dilution
factor of -1 using a micropipette and was placed on the culture media.
Subsequently, one hand was placed outside the cabinet to hold the L-rod
and disinfect it through flaming with the use of the alcohol lamp.
9. The disinfected L-rod was used to spread the liquid on the agar plate until
the surface felt rough.
10. The agar plate was then covered with a transparent lid and labelled
according to the type of the sample and its dilution factor.
11. Following that, the culture media was carried outside and sealed. A section
of the Parafilm M was cut with scissors, separating the polyolefin from the
paraffin wax. The paraffin wax was then applied to the side of the plate and
extended to cover the full side, thereby sealing it.
12. This was conducted nine (9) more times, with two (2) agar plates allocated
for each dilution factor.
13. The culture media were then placed inside the incubator at 37°C and
incubated for 24-48 hours.
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A
Week 3: Assessment and Characterization of the Observed Results
Day 1 - Isolation of Bacteria
1. On November 28, 2024, the third part of the laboratory activity was carried
out after wearing the proper PPE and a quick lecture about the procedures.
2. After assessing the growth mediums, the group decided to isolate bacteria
from the two plates with the dilution factor of -2. These were placed inside
the Biosafety Cabinet along with five (5) new culture media and an
inoculating loop. Outside the cabinet, an alcohol lamp was placed to be
used for sterilizing the loop.
3. To isolate bacteria, the quadrant streak method was utilized. It began with
sterilizing the inoculating loop by placing it over the fire of the alcohol lamp
until the wire became red. The sterilized loop was then cooled down and
used to scrape a small amount of colony from the growth medium. This was
then streaked on half of the culture media or the first quadrant.
Subsequently, the loop was sterilized and cooled down once more.
4. The culture media was rotated at 90° and the second quadrant was
streaked by allowing the inoculating loop to touch the first quadrant for at
least four times before streaking at the empty portion until it reached the
center of the media. Once again, the inoculating loop was sterilized to
ensure the accuracy and integrity of the experimental results.
5. For the third quadrant, the media was rotated again at a right angle, and the
sterilized loop was allowed to touch the streaks from the second quadrant
for only one time, then the streaks were done on the remaining empty
portion of the culture media.
6. The streaked culture media was covered with the transparent lid, labelled
according to the source of bacteria and the member who did the streaks,
and sealed using the Parafilm M.
7. These were then incubated at 37°C and incubated for 24 hours.

Day 2 - Characterization of the Observed Results

1. The following day, the students of BSSE 3A went back to the ParSU-MTL
to characterize the growth mediums by conducting a morphological
observation.
2. Ms. Comal, one of the assistants of the ParSU-MTL gave instructions on
how to characterize the bacteria. With the material provided by Ms. Comal,
the students evaluated their growth mediums by assessing the colonies’
physical characteristics.

IV. Statistical Tool

The number of Colony Forming Units (CFU) per ml is commonly used in

microbiology to quantify the number of viable microorganisms in a liquid
sample. This formula allows for the estimation of the concentration of viable
organisms in the original sample:

CFU/ml = (No. of colonies x Total dilution factor)/Volume of culture plated in ml

Where:
• No. of colonies - This is the number of distinct colonies that grew on the
agar plate after incubation.
• Total dilution factor - This refers to the dilution factor applied to the
sample before plating.
• Volume of culture plated in ml - This is the volume of the liquid culture
that was actually plated on the agar plate, typically measured in milliliters
(ml).
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A

V. Results and Discussion

Sample Name Colony Count CFU/ml

WR1 125 0.0125x105

WR1' 49 0.0049x105

WR2 27 0.027x105

WR2’ 21 0.021x105

WR3 355 3.55x105

WR3’ 0 0

WR4 0 0

WR4’ 0 0

WR5 0 0

WR5’ 0 0
Table 1. Colony-Forming Unit (mg/L)

The results of the study found varying amounts of microbial contamination

in the water samples. Sample WR₃ had the largest microbial load, with a colony
count of 355, representing 3.55 × 10⁵ CFU/mL. This indicate substantial
contamination, signifying that the sample had low water quality or has been
exposed to external pollutants. While, samples WR₃', WR₄, WR₄', WR₅, and WR₅'
lacked detectable microbial activity, with zero colony counts. This indicates either
efficient sanitation or naturally low contamination levels in these water sources.

The study identified a reduction in colony counts in prime-labeled samples,

indicating the efficiency of treatment techniques such filtration or disinfection, with
similar reductions in paired samples WR₁, WR₂, and WR₂'. During the experiment,
the pipette tip accidentally slipped into the culture media during the spreading
method for Sample WR₃, potentially introducing contaminants causing an
unusually high colony count. This could have an effect on the results, requiring
retesting under controlled conditions.

The primary sample WR3 has a high microbial load, possibly due to external
pollutants or water quality issues, while its duplicate sample WR3' has no
detectable colonies. This discrepancy could be due to sampling errors, such as
improper collection or handling of the duplicate sample.

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5

Shape Circular Circular Circular Circular Circular
Margin Entire Even- Entire Entire Entire
Entire
Elevation Raised Flat Raised Convex Raised
Size Punctiform Small Moderate Punctiform Punctiform
Texture Smooth Smooth Smooth Smooth Smooth
Appearance Glistening Glistening Glistening Glistening Glistening
(Shiny) (Shiny) (Shiny) (Shiny) (Shiny)
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A
Pigmentation Nonpigmented Pigmented- Nonpigmented Pigmented- Nonpigmented
- Cream Yellow - Cream Yellow - Cream

Optical Opaque Translucent Opaque Translucent Opaque

Property
Table 2. Morphological Characterization

Consistent circular colony morphology with an entire margin and different

elevations (flat, convex, or elevated) was observed in all five samples. Despite
variations in size, pigmentation, and optical characteristics, all samples had a
smooth texture and a glistening (shiny) look. Samples 1 and 5 were opaque,
punctiform, and nonpigmented (cream), but Sample 3 was moderately sized,
convex, and nonpigmented. The yellow color and translucent optical
characteristics of Samples 2 and 4 distinguished them from the other; Sample 2
was small, while Sample 4 was punctiform. These observations point to minor
changes in colony traits that may reflect variations in their biological or
environmental origins.
The morphological analysis of bacterial colonies in water samples reveals
distinct features, including a circular shape and smooth texture. However,
variations in other attributes suggest different bacterial strains or species. Samples
1, 3, 4, and 5 had an entire margin, while Sample 2 had a flat one. Colony sizes
varied, but all had a shiny appearance. Samples 2 and 4 had pigmentation-yellow
colonies, suggesting different bacterial types. These findings suggest a diversity
of bacterial contamination across water samples, highlighting the need for further
testing and proper water treatment measures.
Due to lack of time, the group was unable to observe microorganisms under
a microscope or perform Gram staining, which is an essential technique for
classifying bacteria based on differences in their cell walls. This obstructed them
from evaluating the morphological features and behavior of the microorganisms,
which is essential determining their biological nature.

VI. Conclusion
Bacterial colonies from a water reservoir sample were effectively
cultured and characterized in this study, revealing notable differences in microbial
load and morphology. The significant level of contamination in WR₃ emphasizes
the need of thorough aseptic procedures and possible concerns to water quality.
While key objectives were met, the lack of Gram staining and microscopic analysis
limited the scope of identification. These results highlight the importance of
thorough microbiological analysis and regular water quality monitoring for
maintaining public health safety.
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A
VII. Documentation
Week 1
(Introduction to Bacteria Culturing and Media Preparation and Demonstration
of Laboratory Equipment)

Week 2
 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A

Week 3 (Day 1 and 2)

 Republic of the Philippines
PARTIDO STATE UNIVERSITY
Camarines Sur
PS-2 BSSE 3A

VIII. References

Guidelines for drinking-water quality. (2022). World Health Organization.

https://www.who.int/publications/i/item/9789240045064

Haneef, J. (2021, June 15). How to Calculate CFU per ml of a Bacterial sample?
In simple 3 steps. Biology Exams 4 U; Blogger.
https://www.biologyexams4u.com/2021/06/how-to-calculate-cfu-per-ml-of.html

How to culture bacteria. (2022, December 12). Retrieved November 30, 2024, from
INTEGRA website: https://www.integra-
biosciences.com/global/en/blog/article/how-culture-
bacteria?fbclid=IwZXh0bgNhZW0CMTEAAR3rg97miA2i41eEMr7HW5S6QAAk3
wPr6iCjqIfnRgd0Kg5cYWR6LtWc5r8_aem_xKGjN9VkT9cH1ZpZLwlEiQ#:~:text=
Bacterial%20culture%20refers%20to%20the,most%20important%20factors%20i
s%20oxygen

Rachael. (2020, April 27). How to See Bacteria on Your Hand (Bacteria Handprint).
Rs’ Science. https://rsscience.com/how-to-see-bacteria-on-your-hand-bacteria-
handprint/

World Health Organization. (2008). Guidelines for drinking-water quality:

Incorporating 1st and 2nd addenda (3rd ed., Vol. 1, Recommendations). World
Health Organization. https://iris.who.int/rest/bitstreams/907844/retrieve

IX. Annex A.

Colony-Forming Units Computation

CFU/ml = (No. of colonies x Total dilution factor)/Volume of culture plated in ml

CFU/ml = (125 x 10 )/10 3 5

= 0.0125 x 10 CFU/ml in sample WR
5
1

CFU/ml = (49 x 10 )/10

3 5
= 0.0049 x 10 CFU/ml in sample WR ’
5
1

CFU/ml = (27 x 10 )/10

3 5
= 0.027 x 10 CFU/ml in sample WR
5
2

CFU/ml = (21 x 10 )/10

3 5
= 0.021 x 10 CFU/ml in sample WR ’
5
2

CFU/ml = (355 x 10 )/10 3 5

= 3.55 x 10 CFU/ml in sample WR
5
3

CFU/ml = (0 x 10 )/10
3 5
= 0 CFU/ml in sample WR ’ 3

CFU/ml = (0 x 10 )/10
3 5
= 0 CFU/ml in sample WR 4

CFU/ml = (0 x 10 )/10
3 5
= 0 CFU/ml in sample WR ’ 4

CFU/ml = (0 x 10 )/10
3 5
= 0 CFU/ml in sample WR 5

CFU/ml = (0 x 10 )/10
3 5
= 0 CFU/ml in sample WR ’ 5

Annex B.
Colony Morphology Chart