AQU 3263 Kultur Plankton

Lab Report 1

Lab Title: Microalgae culture, microalgae cell counting using

haemocytometer, and phytoplankton biology and morphology.

Submitted by: Lee Han S67710

Group: Group 4

Submission Date: 1/5/2024

1. Introduction
Microalgae are a group of microscopic, unicellular, or multicellular microorganisms that can
be found in marine, freshwater, and brackish water. Microalgae are primary producers in water, and
they are also fundamental to the food chain. Microalgae in aquaculture are important because they
function as a source of nutrients, live feed, water quality management, bioremediation and oxygen
production. Example of microalgae cultured in aquaculture are Chlorella spp., Duanaliella spp.,
Nannochloropsis spp., Isochrysis spp., Chaetoceros spp. and Chlorella spp.. The steps involved in
microalgae culture include sampling, preservation, observation, isolation, pure stock culture, mass
culture, and microalgae cell counting. The objectives of this experiment are to learn the entire process
of microalgae culture and to study the biology and morphology of phytoplankton found in the water
sample.
2. Materials and Apparatus
a) Materials: Lugol’s solution, pure stock culture, agar plate, autoclaved sea water, and convey media
(A, B and C).
b) Apparatus: Plankton net, sampling net, YSI meter, parafilm, dropper, inoculation loop, L-shaped
hockey stick, beaker, conical flask, haemocytometer, and compound microscope.
3. Methodology
a) Sampling
In this experiment, water samples were taken from two locations, Site A and Site B, around the UMT
area. Site A was located behind the hatchery of FPSM, while Site B was located at “Raudah UMT”. A
Plankton net was used to trap phytoplankton in each study site, while a YSI meter was used to collect
readings of water parameters such as dissolved oxygen (DO), pH, salinity, and temperature. Next, the
phytoplankton trapped in the Plankton net were transferred into a 500 ml bottle filled with water taken
from the study site. The bottles were labelled with sample A and sample B.

b) Preservation
5ml of Lugol’s solution was added into bottle A and also bottle B. Lugol’s solution, a mixture of
iodine and potassium iodide, is commonly used in microalgae sampling because it can preserve and
stain. For preservation purposes, Lugol’s solution kills the microalgae while preserving their shape.
For staining purposes, Lugol’s solution stains the microalgae cells, making them easier to identify.
c) Observation
One drop of water sample was taken separately from bottles A and B. The morphology of the
phytoplankton was observed using a compound microscope. Any phytoplankton found were recorded
using a phone camera for identification.
d) Isolation
Microalgae of the desired type were selected under the microscope by using sterile equipment. The
selected microalgae were then transferred onto separated agar plates using both the drop plate method
(using a dropper) and the streak plate method (utilizing an inoculation loop and an L-shaped hockey
stick).
e) Pure stock culture
200 mL of autoclaved seawater was measured and placed into a 250 mL conical flask. Conway media
was then added to the flask according to the following formula: solution A = 1.0 mL/L, solution B =
0.5 mL/L, and solution C = 0.1 mL/L. The microalgae grown on the agar plate were carefully scraped
off using a sterile inoculation loop and transferred into the conical flask containing the culture media.
The flask was sealed with parafilm. The microalgae were provided with aeration and lighting at a low
room temperature for one week. Throughout the culture period, the colour of the green water in the
conical flask were recorded everyday by using a phone camera.
f) Mass culture
After one week, the 200 mL of pure stock culture was transferred into a larger conical flask.
Autoclaved seawater was added to the new conical flask until the water level reached 1000mL.
Conway media was then added to the flask according to the following formula: solution A = 1.0
mL/L, solution B = 0.5 mL/L, and solution C = 0.1 mL/L. Aeration, lighting, and suitable temperature
were provided, then the opening of the conical flask was sealed using parafilm.
g) Cell counting
For two weeks, 1 mL of mass culture water was put into a haemocytometer and the number of
microalgae was counted under the microscope once every two days. The result of microalgae
population is recorded in a table and their growth curve was showed in a graph.
4. Result
a) Water parameter (Location A and B)

Location A B
Temperature 29.13°C 28.11°C
pH 7.98 5.96
Salinity 15.14ppt 0ppt
DO 3.35mg/L or 47.5% 9.86mg/L or 26.2%

b) Colour changes of green water

Increased colour intensity

Day 1 Day 2 Day 3 Day 4 Day 5 Day6 Day 7

c) Growth curve of microalgae

Group 3 Group 4

Day of No. of cell/ml Day of No. of cell/ml

culture culture
1 (25/3) 2.80 x 10^5 1 (25/3) 2.34 x 10^5
2 (26/3) 5.06 x 10^5 2 (26/3) 2.94 x 10^5
3 (27/3) 2.88 x 10^5 3 (27/3) 3.0 x 10^5
4 (28/3) 5.58 x 10^5 4 (28/3) 4.18 x 10^5
5 (29/3) 9.08 x 10^5 5 (29/3) 4.46 x 10^5
6 (30/3) 5.56 x 10^5 6 (30/3) 4.98 x 10^5
7 (31/3) 6.30 x 10^5 7 (31/3) 4.24 x 10^5
8 (1/4) 9.98 x 10^5 8 (1/4) 4.08×10^5
9 (2/4) 10.06 x 10^5 9 (2/4) 4.26 x 10^5
10 (3/4) 3.0 x 10 ^5 10 (3/4) 4.48 x 10^5
11 (4/4) - 11 (4/4) 5.58 x 10^5

*Example of calculation for no. of cell/ml (example from group 4, day 5)

Total number of microalgae counted in 5 plots = 52+35+51+47+38 = 223
223 4
No. of cell/ml = ×1 0 = 4.46 x 10^5
5
 Population density of microalage against day of culture
12
Population density of microalgae (x 10^5)

10

0
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11
Day of culture

Group 3 Group 4

d) Types of phytoplankton found in location A and B

Location A

Chlamydomonas spp. Coelastrella spp.

Cyclotella spp. Chaetoceros spp.

Tetmomerus spp.
Location B

Diatom

5. Discussion
In this experiment, the location A and location B have different water parameters. Based on
the results recorded, location A is a brackish water environment (15.14ppt), and location B is a
freshwater environment (0ppt). The concentration of dissolved oxygen in location B (9.86mg/L) is
higher than that in location A, this may be caused by two factor, temperature and salinity. When the
temperature is higher, the oxygen molecules in the water gain more kinetic energy and will tend to
leave the water and enter the air; when the temperature is lower, the oxygen molecules have lesser
kinetic energy, therefore it is more difficult to leave the water and enter the air. In higher salinity, the
salt in the water will compete with the oxygen to bind with the water molecule, thus reducing oxygen
solubility in higher salinity environment. For pH, location B is an acidic environment whereas
location A is a slightly alkalic environment.
Next, the second result of this experiment is colour changes of green water. In overall, we can
see that the green colour intensity increases day by day. Although there is a drop in green colour after
day 4, but it still continues to increase until day 7.
For the population density of microalgae against day of culture, a growth curve graph is used
to compared the results from group 3 and group 4. In most of the time, the population density of
microalgae in group 3 is higher than that in group 4, except for day 3 and day 10. Also, the maximum
population density of microalgae in group which is 3 is 10.06 x 10^5/ml, is much higher than the
maximum population density of microalgae in group 4 which is 5.58 x 10 ^5. The reason why the
population density of microalgae in group 3 is higher than that in group 4 may be due to the group 3
provides a better mixing and aeration to the microalgae, or may be due to ciliate contamination in
group 4. An efficient mixing and aeration can provide sufficient oxygen and distribute the nutrient
evenly to the microalgae, while ciliate contamination will decrease microalgae population because
they hunt for microalgae as food.
Lastly, for the microalgae identification, in location B, there is only one microalga
successfully identified, which is the diatom., whereas in location A, there are 5 microalgae identified,
they are Chlamydomonas spp., Coelastrella spp., Cyclostella spp., Chaetoceros spp., and Tetmemorus
spp.

6. Conclusion
In conclusion, location A is a brackish environment, while location B is a freshwater
environment. Aeration, temperature, nutrients and lightning are the major factors to success culture of
microalgae, if there is no ciliate contamination occur. When the growth rate is higher than the death
rate, the population density of microalgae will increase; when the death rate exceeds the growth rate,
the population density of microalgae will decrease. Lastly, due to limited knowledge, just a few type
of microalgae have been identified in this experiment, but actually there are still a lot more microalgae
in the location A and B that is not yet identified, more effort are need to put in future to identify more
microalgae in the selected locations.

7. References

1. Chaetoceros sp. - The Official Website of Sarawak Biodiversity Centre. (n.d.). Www.sbc.org.my.
Retrieved April 27, 2024, from https://www.sbc.org.my/component/content/article/865-
chaetoceros?catid=187&Itemid=201#:~:text=A%20centric%20diatom%20with%20very

2. Blanken, W., Postma, P. R., de Winter, L., Wijffels, R. H., & Janssen, M. (2016). Predicting microalgae
growth. Algal Research, 14, 28–38. https://doi.org/10.1016/j.algal.2015.12.020

3. RAMLEE, A., W. RASDI, N., ABD WAHID, M. E., & JUSOH, M. (2021). MICROALGAE AND THE
FACTORS INVOLVED IN SUCCESSFUL PROPAGATION FOR MASS PRODUCTION. JOURNAL of
SUSTAINABILITY SCIENCE and MANAGEMENT, 16(3), 21–42.
https://doi.org/10.46754/jssm.2021.04.003

4. What Is The Relationship Between Dissolved Oxygen And Salinity? (2022, August 23). Atlas
Scientific. https://atlas-scientific.com/blog/dissolved-oxygen-and-salinity/#:~:text=Dissolved
%20oxygen%20and%20salinity%20are