Final Proposal Rb09

8/4/2019 Final Proposal Rb09

http://slidepdf.com/reader/full/final-proposal-rb09 1/45

The Use of Ultrasonic Waves as an AlternativeMethod in Mitigating Algal Blooms

By

Madrid, Ludhovik Luiz B.

Medrana, Micholo Lanz B.Morales, Justin Marius D.

Submitted to the Faculty of thePhilippine Science High School – Main Campus

in partial fulfillment of the requirements for

Science and Technology Research 1

March 2011



ABSTRACT

One type of Harmful algal bloom (HAB) may cause the discoloration of seawater

caused by a dense population of dinoflagellates. However, not all types of HABs can cause

water discoloration. HABs have been recurring in Philippine waters since 1988 and are

notable for their disruption to the marine ecosystem and their negative health and economic

effects. The aim of this project is to determine the effects of time exposure of ultrasound

waves on Pyrodinium bahamense var. compressum to know whether these variables would

lead to inhibition of growth or complete elimination of the organisms.

From an initial culture of the dinoflagellate P. bahamense, 24 Erlenmeyer flasks

containing a 100 mL culture solution will be prepared and divided into 12 separate

experiment groups of 2 flasks each. Each experiment group will test the effect of a same

frequency (1 MHz) on a sample exposed for different time spans. Control samples will

receive no treatment. After each treatment is performed, a cell count will be taken of the

sample using a Sedgewick-Rafter slide and recorded. The results of each experiment group

will be statistically analyzed to determine any significance between the treatments and the

control. This analysis will help determine the optimum exposure time for inhibition of

growth or complete elimination of the organism.



i

APPROVAL SHEET

This research work entitled, “The Use of Ultrasonic Waves as an Alternative Method

in Mitigating Algal Blooms” by Madrid, Ludhovik Luiz B., Medrana, Micholo Lanz B., and

Morales, Justin Marius D., presented to the Faculty of the Philippine Science High School –

Main Campus in partial fulfillment of the requirements in Science & Technology Research 1,

is hereby accepted.

____________________________________

Dr. Jessamyn Marie O. Yazon, Ph.D.

Research Adviser



ii

ACKNOWLEDGMENTS

First, we would like to thank ourselves, for doing our best in working on this

research, and in winning the oral defense in YMSAT. We would like to thank our classmates,

for coming along with us while we “cram” our STR requirements. We would like to thank

our research teacher, Ma’am Yazon, for reviewing on our work, and pointing out mistakes

and improvement on our STR requirements. We would also like to thank some of our

teachers: Ma’am Chupungco, Ma’am Docto, Ma’am Buenafe, and Sir Tan for giving us

advice during the oral defense and Sir Talaue for giving us the research journals that we

cannot access. We would also like to thank the people of MSI (Sir Gar ry, Ate Jenelle, Ma’am

Lita, Dr. Azanza) for helping us in our research, Williard Jose (III-Be), Dr. Jose, and Dr.

Escoto, who helped us find a contact person who has an ultrasound machine, and Mr.

Publico, for letting us borrow his ultrasound cleaner.

Last but not the least; we would like to thank God, for giving us the wisdom, strength,

and inspiration to finish our research, and for giving us hope whenever we had problems in

STR.



iii

TABLE OF CONTENTS

Page

Approval Sheet i

Acknowledgments ii

Table of Contents iii

List of Tables v

List of Figures vi

I. Introduction

A. Background of the Study 1

B. Statement of the Problem 2

C. Significance of the Study 2

D. Scope and Limitations 3

II. Review of Related Literature

A.

Harmful Algal Bloom (HAB) 5

B. Algal Culture 9

C. Ultrasonic Principles 10

III. Materials and Methods

A. Procurement of Algal Solutions and Other Materials 14

B. Preparation and Sterilization of Culture Flasks 14

C. Preparation of Algal Solutions 15

D. Initial Cell Counting 19

E. Setting up of Ultrasonic Cleaner 20



iv

F. Exposure of Algae to Ultrasound and Final Cell Counting 21

G. Computation, Graphing, and Tabulation of Collected Data 21

H. Statistical Tests (ANOVA and T-test) 22

IV. Bibliography 23

Appendices

A. Summary of Materials and Methods 26

B. Formulas, Tables, and Graphs needed for Data and Analysis 27

C. Risk Assessment 29

D.

Task List 32

E. Materials Sourcing and Budgeting 34

F. Gantt Chart 36

G. Network Chart 36



v

LIST OF TABLES

Table Title Page

5.2.1 Table for Analysis of Variance (ANOVA) 27

5.2.2 Table Comparing Initial and Final Cell Density for each Treatment 27

5.2.3 Table for Correlated T-Test 27

5.2.4 Initial, Final, and Change of Cell Densities of Pyrodinium bahamense var.

compressum cultures in Concentration of Algae vs. Time of Exposure

28

5.3 Risk Assessment Table 29

5.4 Task List of the Methods in the Research Study 32

5.5.1 Table of Materials needed for Experiment and their Costs 34

5.5.2 Table of Transportation and Electricity Consumption 34



vi

LIST OF FIGURES

Figure Title Page

3.2.1 Erlenmeyer flasks obtained in MSI (left picture), and Erlenmeyer flasks were

cotton seal (right picture)

14

3.2.2 Water filterer connected to a container for filtered seawater (left picture).

Erlenmeyer flask filled with filtered seawater (middle picture). Inside the

autoclave are Erlenmeyer flasks with filtered seawater and seals covered with

aluminum (right picture)

15

3.3.1 Things needed for preparation of algal solutions. From left to right: stock

culture, alcohol lamp, single channel pipette, autoclaved Erlenmeyer flasks

with filtered seawater, and F/2 medium.

16

3.3.2 Aseptic methods for sterilization purposes. Erlenmeyer flask’s mouth was

heated (left), and F/2 container’s mouth was also heated (right)

17

3.3.3 Transferring of F/2 medium from container to Erlenmeyer flask 17

3.3.4 Transferring stock culture to Erlenmeyer flask near the open flame (aseptic

method)

17

3.3.5 All 24 flaks kept near a light source 18

3.4.1 Things needed for cell counting (from left to right): Eppendorf tubes in

orange case, culture flask, and single channel pipette.

19

3.4.2. Researchers working on cell count while looking in the microscope 20

3.5. Ultrasonic Cleaner Set-up 20

5.1. Process Flowchart for The Research Study 26



vii

5.6 Gantt Chart with Expected Dates of Work 36

5.7 Network Chart of the Research Study 36



1

I. INTRODUCTION

A. Background of the Study

A Harmful Algal Blooms (HAB) is a discoloration of seawater caused by a dense

population of a given species of dinoflagellate (Villao, 1988). In some cases, blooms are

accompanied by toxins which, when ingested by surrounding marine life, are capable of

poisoning them or their predators. In the Philippines, especially in the waters of Manila Bay,

HABs are caused by the dinoflagellate Pyrodinium bahamense var. compressum, which have

recurred here since 1988 (Azanza, 1997).

HABs have a direct effect on the coast and its underlying areas. Examples of such

effects include impacts like fishkill (which is caused either by poisoning or oxygen

depletion) and Paralytic Shellfish Poisoning (PSP), a disease in humans caused by the

consumption of tainted shellfish resulting in either partial or complete paralysis of the body

(Villao, 1988). It is these negative effects of HABs that have motivated scientists in multiple

related fields to devote their research efforts on finding methods to reduce HABs.

One method that has not been attempted yet (as far as the group's preliminary

research went) is the use of ultrasound to control HABs. Ultrasound has proven successful as

a method of controlling freshwater algal blooms (caused by cyanobacteria) in agricultural

areas, both locally and overseas (Song, et al., 2005). Removal of cyanobacteria in these

environments is needed due to their oxygen depleting effects with regards to agriculture and

their toxic nature, especially in sources of potable water (Falconer, 1999).



2

The use of ultrasound to control freshwater algal blooms employs the concept of microscopic

cavitation resulting from high frequencies, resulting in biological disintegration. Stemming

from this concept, the research will see whether the use of these ultrasound waves can be

applied in the control of HAB's, bringing us to the statement of the problem.

B. Statement of the Problem

This study aims to test whether the exposure of harmful algal blooms (HABs),

specifically those caused by the blooms of the dinoflagellate Pyrodinium bahamense var.

compressum, to ultrasound waves would result in the mitigation or reduction of these

blooms. Assuming this is feasible, the secondary objective of this study is to determine the

ideal length of time it would take for ultrasound exposure to effectively control HABs. The

dinoflagellate’s population size and concentration are aspects that will be observed and

measured during the course of this study. This study also attempts to see whether the

ultrasound transducer to be used in the experiment can easily be deployed on the field,

without necessarily experimenting outside of a controlled laboratory setup. Accomplishing

these goals will lead to the real life problem, which is to find a method of controlling HABs.

C. Significance of the Study

In the research of HAB's, the field of studying Harmful Algal Bloom control has been

progressing far too slowly. Though there are new methods of harmful algal bloom control

(use of chemicals, genetically engineered species, clay flocculation), these methods have

harmful effects on the environment and biological life other than HAB's (Anderson, 2005).



3

With the incremental develop of the industry and economy, the HAB (Harmful Algal Bloom)

problems gradually become a cosmopolitan marine disaster, which endangers the health of

the people and the fishery ecosystem (Jinhui, 2005). Fisheries in the Philippines suffer from

fish kills caused by these HAB’s, and the lives of seafaring Filipinos are also more at risk.

Using the methods for HAB control that were mentioned above would only worsen the

problem, highlighting the need for a practical method that will not bring harm to species

other than HABs. This problem is solved by the use of ultrasound to control populations of

HABs, which would also reduce the number of fish kills. This would in turn cause an

increase of the fish supply of our country and decrease the number of fatalities caused by

PSP. Thus, solving this problem would help our country’s poverty problems and its

economic status.

D. Scope and Limitations

The study will be done over a course of around one and a half months. Cultures of

Pyrodinium bahamense var. compressum from the Marine Science Institute (MSI) wasused

in the research study. Twelve groups consisting of two replicates each will be exposed to

different lengths of time. The frequency of ultrasound that was used is 1 MHz. The time of

exposure (independent variable) will vary from each treatment, ranging from 1 hour to 6

hours. The experiment will find out if the optical density, the dependent variable, will

increase, decrease, or remained unchanged. Other than the frequency of the ultrasound, time

of exposure to light, volume of solution, amount of nutrients, salinity, and temperature will

be considered as controlled variables. This study aims to cover the control of the population

of HABs and does not deal with the complete elimination of HABs.



4

This study is completely different from the study entitled Using Ultrasound to

Control Algal Blooms which was conducted by Dr. Carl Howard and a team of researchers

from the University of Adelaide. The research of the aforementioned team deals with the

effects of ultrasound to blooms of blue-green algae, whereas this research tests if various

time exposures to ultrasound have an effect on HABs.



5

II. REVIEW OF RELATED LITERATURE

A. Harmful Algal Bloom (HAB)

What is a HAB?

An HAB, commonly known as “red tide”, is the discoloration of a body of water

caused by massive populations of coastal dinoflagellates (Villao, 1988). Commonly

distributed in the tropical Indo-Pacific region, HABs are predominant in numerous countries

including the Philippines (Busine, et al., 2003).

Factors affecting HAB growth

Dinoflagellates within HABs prefer high surface-water temperatures and high light

intensities, though this does not mean that HABs only occur in tropical areas. HABs occur in

hot, calm weather because surface temperatures warm up even in normally cool areas

(Badylak, et al., 2004; Maclean, 1977).

Light wind intensity moves the bloom near the coast, whereas strong wind intensity

aids in the swimming of dinoflagellates to the surface. Storms on the other hand, even with

strong wind intensity, disperse the HABs. Storms also result in the death of dinoflagellates

and can prevent the development of red tides (Pollingher & Zenel, 1981).

Red tides usually occur after an upwelling has stopped, but the nutrients brought to

the surface do not appear to be the direct cause of these blooms (Grindley & Nel, 1970). The

overloading of nutrients to bodies of water, or eutrophication, gives favorable conditions to

HABs. The presence of HABs, however, has proved to be detrimental to other organisms in

the environment (Nybakken, 1982).

Effects of HAB to marine and human environment



6

HABs have been a significant global and national concern due to their negative public

health and or/economic effects (Fernandez & Ricafrente, 2010). Red tides deplete the

nutrients needed for other marine organisms to survive. They also cause oligotrophication,

which is the abundance of toxic materials in bodies of water. HABs have the ability to

produce very lethal toxins like saxitoxin, Neo-saxitoxin and decarbamoyl, which can cause

the death of marine organisms. These toxins can also accumulate in some animals, and can

result in Paralytic Shellfish Poisoning (PSP) in humans if these animals are eaten. Symptoms

of PSP include paralysis, vomiting, shortness of breath and other difficulties, and can result

in death through respiratory failure (Busine, et al., 2003).

HABs have economic effects on multiple sectors of society. Fish kills caused by

HABs reduce fish supply, while shellfish bans imposed by local authorities caused losses in

the fishing industry, pose problems to international trade, and result in underemployment of

industries (Bajarias, et al., 2003).

Methods of HAB management and monitoring in the Philippines

Current methods of HAB management are limited to detection and analysis of the

areas affected by a bloom, including the implementation of bans on shellfish and similar

products affected, and an information campaign by various government agencies. This

management method has been done since 1984, and is inefficient in the sense that very little

is done to control the bloom physically, and simply involves waiting for it to fade out

(Bajarias, et al., 2006). Other physical means, such as domoic acid treatment, of controlling

and eliminating HABs are still being tested out by various government agencies such as the

PCAMRD (Fernandez & Ricafrente, 2010).



7

However, the Philippine government keeps on monitoring seas that are known to

have past cases of red tide. The Department of Health (DOH), and Bureau of Food and Drugs

(BFAD) keeps on acquiring samples of seawater from the seawater to obtain quantitative

analysis of cell density and toxin concentration. Aerial surveillance is also monitored by the

Department of Agriculture (DOA), and Philippine Air Force, to see if there is any

discoloration of the sea’s surface (Busine, et al., 2003).

Methods of HAB management and monitoring outside Philippines

Countries have advanced technologies that the Philippine lacks, and one of them is

the satellite monitoring system (Bajarias et al., 2003). Countries such as China, Norway,

Canada, Japan, and USA use remote sensing satellites to detect HABs. With the use of

satellites, aerial surveillance is much easier (Andersen, et al., 2001; Busine, et al., 2003).

They also train fishermen on how to detect HABs and collect HAB samples with the use of

buoys, lighthouses, and plankton nets. Also, the marine farms are more advanced than in the

Philippines; their fish and mussel farms contain laboratories that separate the poisoned

marine organisms from the healthy ones (Anderson, et al., 2001; Bajarias, et al., 2003).

These countries also developed ways on how to control the population of algae, but

these have limitations: adding of chemicals, flocculation, and biological manipulation.

Adding of chemicals started when the US government added copper sulfate to seas

with HABs near Florida. Adding copper sulfate was effective, but after weeks, the HAB

reestablished itself, killing more fish along the shores of Florida, and calm wind currents also

carried part of the bloom to other seas of United States. They also used other chemicals such

as ozone and aponin, but these have negative effects in marine life other than harmful algae.

Flocculants, substances that capture suspend particles until they become heavy and fall as



8

sediments, were tested in Japan and were found to be effective against HABs, but it is very

expensive. Japanese researchers also used the addition of viruses to destroy the algae, and

bacteria, dinoflagellates, and other zooplankton to compete with the algae. It was effective,

but it also massive bivalve and fish kills (Andersen, et al., 2003; Newcombe, 2009).

Pyrodinium bahamense var. compressum

In the Philippines, Pyrodinium bahamense var. compressum has been identified as the

main organism responsible for HAB outbreaks in Manila Bay since 1988. P. bahamense

forms its “blooms” by cell division during their vegetative stage, and is easily grown in a

laboratory setup, given the proper conditions. P. bahamense has a growth rate of one cell

division every three days, and produces a planozygote in later stages of its bloom because of

a union of its gametes. A non-motile hypnozygote is then formed, which brings the organism

into dormancy for about three to four months, unless optimal conditions of salinity,

temperature, and light intensity are established. Once these conditions are met, the

hypnozygotes then germinate via meiosis and then binary fission, in turn initiating a new

bloom (Azanza, 1997).

Blooms of P. bahamense are dominant in Bolinao, Masinloc, Manila, Palawan,

Camiguin, Surigao Province, Leyte, and Samar. These areas are reportedly to have massive

fish kills, especially in the Manila Bay. However, the effects of El Nino and La Nina disrupt

the normal growth pattern of the algae, so fish kills in these areas do not happen at the same

time (Azanza, 1997; Bajarias, et al., 2003; Busine, et al., 2003).

Saxitoxins are considered the most potent neurotoxin found in dinoflagellate blooms

(Busine, et. al., 2003). Aside from Saxitoxin, Pyrodinium also produces other types of

neurotoxins, particularly Neo-saxitoxin, decarbamoyl, gonyautoxin-5 and gonyautoxin-6



9

(Busine, et. al., 2003; Corrales, 1991). With these toxins, it is necessary to note that proper

algae culturing protocols would need to be implemented in this research, for the safety of the

researcher (Azanza, 1997).

B. Algal Culture

Factors considered in growing algae

Like all organisms, certain factors should be considered in growing algae. As with all

plants, algae must have sufficient nutrients to support growth, and factors, such as light,

temperature, salinity, seawater quality, mixing and cleanliness will all need to be kept

constant to ensure favorable conditions for algal growth (Food and Agriculture Organization

of the United Nations, 2007; Hallegraeff et al., 1995).

Techniques in growing algae

Aseptic method is strictly imposed when culturing microorganisms. It prevents

addition of unwanted microorganisms. While adding nutrients, or the stock culture, you must

make sure you heat the mouth of the container, or transfer the necessary components near an

open flame (Food and Agriculture Organization of the United Nations, 2007).

In an algal culture system, a stock culture should be maintained. These stock cultures

provide the reservoir of algal cells from which to start the larger-scale cultures used for

feeding. In preparing the replicate culture flasks, use the pipette to transfer a small sample of

the stock culture to the flasks. Use a separate pipette for transferring the nutrients. For

dinoflagellates, their nutrient is F/2 medium, a mixture of vitamins, nitrates, phosphates,

silicates, and other minerals (Food and Agriculture Organization of the United Nations,

2007).



10

Algal cultures must be monitored daily to detect if there are dead species, cell

aggregation or clumping, or contaminants. If a culture contains multiple clumped cells, cells

with cell walls broken, more than one species present, or is contaminated with foreign

bodies, the culture should be discarded (Food and Agriculture Organization of the United

Nations, 2007).

Cell counting methods

Monitoring of the algal cultures will also involve a cell count, using either a

haemocytometer or a Sedgewick-Rafter counting slide.

A haemocytometer is widely used by researches because of its easy instructions, its

lightweight use. However, haemocytometer is not suitable for algae larger than 45 microns,

such as P. bahamense. Haemocytometers also rely on calculations and estimation, which can

be inaccurate (Karlson, et al., 2010; Tech Note, 2004).

This research study proposes the Sedgewick-Rafter counting slide, a traditional

counting method. It contains a 20 by 50 grid of 1 mm2 squares. It is recommended with cells

of large size and population. It does not need calculations, but the counting is manual and

time-consuming (Karlson et al., 2010).

Aside from establishing principles and culturing techniques of HABs, this research

will also note the scientific concept of ultrasound and its effects on biological and chemical

materials.

C. Ultrasonic Principles

Basic Ultrasonic Principles

Ultrasound is defined as a high frequency that is above 20 kHz, above the audible

range. Ultrasound waves require an elastic medium, such as solids or liquids, for



11

transmission, operated by stressing the medium itself. Its wavelengths are small compared to

audible waves, and it is said to have a “high frequency” relative to the audible range of

humans (Agarwal, 2005).

The most common methods of ultrasonic examination utilize either longitudinal

waves or shear waves, and other forms of sound propagation exist, including surface waves

and Lamb waves. Surface waves are characterized by elliptical particle motions and are

bound to the surface of a material, while Lamb waves are characterized by complex

vibrations in materials where its thickness is less than the ultrasound wave induced to it

(Olympus, 2006).

Ultrasound can be classified into two types: low frequency ultrasound, and high

frequency ultrasound. Low frequency ultrasound ranges from 20 kHz to 1 MHz, and high

frequency ultrasound ranges from 1 MHz onwards (Van Iersel, 2008).

Biological Effects of Ultrasound

There are ways in which ultrasound can produce biological effects such as cavitation,

microstreaming, and heating (Chudleigh & Thilaganathan, 2004). Cavitation is the

ultrasonically induced activity occurring in a liquid or liquid-like material that contains

bubbles or pockets containing gas or vapor (O’Brien, 2007), and can rupture the cell

membranes of microorganisms (Li, 2009). Microstreaming is the formation of small local

fluid circulations, and can be intra- or extracellular. Microstreaming can cause the same

effects to those of cavitation. Heating is caused by the absorption of the ultrasound wave by

organisms, and it can cause internal injuries to the cell (Chudleigh & Thilaganathan, 2004).

Low frequency ultrasound is enough to produce cavitation, microstreaming, and heating in

aquatic environments (O’Brien, 2007; Van Iersel, 2008).



12

In tissues of macroscopic animals, high frequency ultrasound can only cause

abrasions in the tissues. Heating and not cavitation is the cause of tissue damage in animals.

As frequency increases, absorption of ultrasound waves increases. As the tissue absorbs the

ultrasound waves, their vibration causes to increase the internal energy of the tissue, which

creates heat (Carstensen, et al., 1974).

Chemical Effects of Ultrasound

In cavitation, the induced activity of bubbles or pockets can grind insoluble

substances in liquid. These insoluble substances will become smaller until it suspends in the

liquid medium (Li, 2009). Ultrasound can also decompose or transform organic molecules,

such as chlorophyll and saxitoxin, into smaller and useless ones. Ultrasound increases the

energy needed to break the bonds between atoms, until the energy reaches the activation

energy, the minimum energy required to have a chemical reaction. Once the organic

molecule changed its form, it can no longer function properly (Emery, et al., 2005; Van

Iersel, 2008). If the harmful algae lacked the necessary compounds needed for

photosynthesis, it can no longer survive (Andersen, et al., 2003).

C. Transducer, the Ultrasound Machine

What is the transducer?

The device that both generates the ultrasound and detects the returning echoes is the

transducer. Transducers are made of materials that exhibit piezoelectricity, which is “electric

polarity due to pressure especially in a crystalline substance” (Fleischer, et al., 1991). In

ultrasonics, the piezoelectric material is the actual transducer because “it converts ultrasound

into electric energy and vice-versa. When voltage is applied, the transducer will expand and

contract, creating ultrasound” (Chudleigh & Thilaganathan, 2004; Sherman & Butler, 2007).



13

Main Components of Transducers

There are three main components of transducers, namely the active element, the

backing, and the wear plate. The active element is the piezoelectric material. The backing

acts as an energy conserver by absorbing the energy radiating from the active element.

Finally, the wear plate protects the transducer element from the surroundings (Fleischer, et

al., 1991; Olympus, 2006).

Sonicators

A sonicator is a device used to break open cells using ultrasound waves, a method

called sonication. Sonicators make use of the properties of high frequency ultrasound to

disrupt the cell wall of an organism. Sonication is employed in most studies for extraction of

enzymes within a cell, with insoluble materials separated by centrifugation (Madison Area

Technical College, 2005).



14

III. MATERIALS AND METHODS

A. Procurement of Algal Solutions and other materials

A stock culture of Pyrodinium bahamense var. compressum was obtained from the

Marine Science Institute (MSI) at the University of the Philippines (UP) – Diliman Campus.

An ultrasonic cleaner was obtained from the I-MAT Pro Company. The Sedgewick-

Rafter counting slide, materials for the construction of the culture tank and all other

laboratory equipment (stirring rod, pipette, waste bottles, Erlenmeyer flasks, reagent bottles,

F/2 media, microscope, autoclave, Lugol’s Iodine, Eppendorf Tubes, etc.) was obtained from

the MSI. Common materials for cleaning (at the end of the experiment) were bought from

public markets.

B. Preparation and Sterilization of Culture Flasks

Twenty-four (24) 125 mL Erlenmeyer flasks were used in the experiment. A seal for

the flasks were made by rolling a thick sheet of cotton. The roll of cotton was inserted in the

hole of the flask to check if it fits. Otherwise, a portion of cotton must be removed. The

process must be repeated several times until the cotton tightly fits in the hole. Then, the

cotton was wrapped with cheesecloth and twisted to form a handle in the top. The handle was

sealed with masking tape.

Figure 3.2.1. Erlenmeyer flasks obtained in MSI (left picture), and Erlenmeyer flasks were

cotton seal (right picture).



15

32 ppt of seawater was filtered by using a water filterer. Seawater must be filtered to

remove unwanted substances (other than salts) (Karlson, et.al, 2010). The filtered seawater

was transferred in a large flask, and each Erlenmeyer flask was filled with 50 mL of filtered

seawater. Then, the seals were covered with aluminum foil.

Figure 3.2.2 Water filterer connected to a container for filtered seawater (left picture).

Erlenmeyer flask filled with filtered seawater (middle picture). Inside the autoclave are

Erlenmeyer flasks with filtered seawater and seals covered with aluminum (right picture).

Autoclaving is necessary to remove unwanted microorganisms inside and outside of

the flask (FAO, 2007). All flasks were placed inside an autoclave for 1 hour and after

autoclaving; they were cooled for the day.

C. Preparation of Algal Solutions

The preparation of algal solutions was executed in a secure, temperature-controlled

room. All stock cultures must be kept in a temperature-controlled room, to control (not alter)

the growth pattern of the algae (Food and Agriculture Organization of the United Nations,

2007; Karlson, et.al, 2010). Aseptic method was used to prevent contamination of unwanted

organisms in the flasks (Food and Agriculture Organization of the United Nations, 2007).



16

Figure 3.3.1 Things needed for preparation of algal solutions. From left to right: stock

culture, alcohol lamp, single channel pipette, autoclaved Erlenmeyer flasks with filtered

seawater, and F/2 medium.

F/2 medium must be added to each flask before adding the algae. F/2 medium is a

broth for the algae’s nutrients. Without F/2, the algae will not survive (Food and Agriculture

Organization of the United Nations, 2007). Before adding the F/2, each mouth of the F/2

container and the flask was heated by an alcohol lamp (aseptic methods). A ratio of 1 L of

algal solution to 2 mL of F/2 was used, since this is the protocol MSI imposed on growing P.

bahamense var. compressum. Each flask contained 100 mL of algal solution, so 0.2 mL, or

200 microliters of F/2 was added, by using a single channel pipette. After each flask has been

added with F/2 medium, the mouth of the stock container and the flask was heated, and 50

mL of P. bahamense var. compressum was added, until all 24 flasks were filled. After this,

all flasks were randomized and labeled acc. to their assigned treatment. There were 1 control

and 11 treatments, and each group has two flasks.



17

Figure 3.3.2. Aseptic methods for sterilization purposes. Erlenmeyer flask’s mouth was

heated (left), and F/2 container’s mouth was heated (right)

Figure 3.3.3. Transferring of F/2 medium from container to Erlenmeyer flask.

Figure 3.3.4. Transferring stock culture to Erlenmeyer flask near the open flame (aseptic

method).



18

The algae will die if there is no light because of absence of photosynthesis

(Hallegraeff et al., 1995). All flasks are kept in the corner of the room near a light source.

Salinity, temperature, light intensity, and wind intensity are the factors to be

considered in the habitat of P. bahamense (Hallegraeff et al., 1995). Salinity was kept

constant by adding the same amount of seawater (in the same concentration of 32 ppt). Since

the flasks are kept in a temperature-controlled room, controlling temperature is not much of

problem. The algae are inside the flasks, which does not have air disturbance. Light intensity

was kept constant by the MSI staff monitoring the time of using the light source in the room.

Figure 3.3.5. All 24 flaks kept near a light source.



19

D. Initial Cell Counting

Figure 3.4.1. Things needed for cell counting (from left to right): Eppendorf tubes in orange

case, culture flask, and single channel pipette.

The number of cells in the cleaner tank was counted by using a 20 x 50 Sedgewick-

Rafter counting slide before exposing them in ultrasound waves. A sample of algal solution

was be obtained by using a single channel pipette, and it must be preserved in Lugol’s Iodine

before counting (Karlson, et al., 2010). 1 mL of the solution was obtained at it was

transferred in an Eppendorf tube. Two 1 mL samples were obtained for each flask, because

the counting consists of two trials. Then, each tube was added with 1 drop o f Lugol’s Iodine

by using a dropper. Adding the Lugol’s Iodine was done outside the room since its fumes can

kill the algae (Karlson, et al., 2010).

Then, the 1 mL solution was transferred, by using a different dropper, to the cover

glass of the counting slide. The cover glass must be slowly swung so it completely covers the

solution. Careful alignment of the cover glass will stop air bubbles from proliferating into the

sample and will ensure that the solution is completely spread in the cover glass. If the

solution is completely spread, the slide is ready for counting.



20

Figure 3.4.2. Researchers working on cell count while looking in the microscope.

The cover glass of the counting slide was viewed in a microscope using a 10x objective. A

counter was used to aid the researcher in counting cells. While counting, if the researcher

sees “x” cells, he will click the counter “x” times. All 1000 squares wer e checked and the

number of cells was recorded. For faster counting, the squares in the slide were scanned in a

zigzag formation (Karlson et al., 2010).

E. Setting up of ultrasonic cleaner

Figure 3.5. Ultrasonic Cleaner set-up.



21

The ultrasonic cleaner served as the culture tank of the experiment, and it was filled

with tap water. Sound waves can pass through solids and liquids, therefore algae can still be

exposed through ultrasound waves (Olympus, 2006). Pyrodinium bahamense var.

compressum can survive in subtle amount of air (Azanza, 1995), so there is no need of

oxygen/carbon dioxide tanks.

2 flasks were placed inside the ultrasonic cleaner. Only the inside of the cleaner must

be wet, and the level of the water must not rise beyond the mark inside the cleaner, to prevent

electrocution (Lee, 2004). If the level of the water goes up beyond the mark, the flasks were

discarded and removed some of the water, until the water level is exactly at the mark.

F. Exposure of Algae to Ultrasound, and Final Cell Counting

After setting up the ultrasonic cleaner, it was switched on. Each treatment was

applied with the same amount of frequency (1 MHz), and with varying amount of time. The

first treatment was exposed with 1 hour of ultrasound. Each succeeding treatment was

exposed with an additional 30 minutes of exposure (90 minutes for the 2nd treatment, 120

minutes for the 3rd, and so on). A countdown timer was used to monitor the time of exposure

for each treatment. At the end of each treatment, the flasks were taken out and the same

process (two trials) was done for counting the algae. After finishing all treatments, the water

inside the cleaner was removed. The cleaner was wiped with tissue until its dry, and it was

kept back in its container.

G. Computation, Graphing, and Tabulation of Collected Data

All data computed or obtained before were presented into tables and graphs using

Microsoft Excel. There will be 12 tables, and each table is assigned for one treatment. The



22

table will consist of the initial cell density and final cell density of the treatment. Then, each

table will be interpreted by using line graphs.

H. Statistical Tests (ANOVA and T-Test)

Analysis of Variance (ANOVA) will be used, with a level of significance of 0.05, to

find out if there is a significant difference between the change of cell densities of each

treatment (alternative hypothesis), or if there is no significant difference (null hypothesis). A

t-test will be used, for each treatment with a level of significance of 0.05, to find out if there

is a significant difference between the initial and final cell densities of the treatment

(alternative hypothesis), or if there is no significant difference (null hypothesis).



23

IV. BIBLIOGRAPHY

Agarwal, S. K. (2005). Advanced biophysics. New Delhi: APH Publishing. (Agarwal, 2005)

Anderson, M., Andersen, P., Bricelj, V.M., Cullen, J., & Rensel, J. (2003). Monitoring and

management strategies for harmful algal blooms in coastal waters. Paris: UNESCO.

Azanza, M.P., Azanza, R.V., & Ventura, S. (2003). Varied assays for PSP toxins inheatshocked Philippine green mussels (Perna viridis). Journal of food safety, 23, 249-

259.

Azanza, R.V. (1997). Contributions to the understanding of the bloom dynamics of P.

bahamense. Science Diliman, 9(1-2), 1-6.

Azanza, R.V., & Hall, S. (1993). Isolation and culture of Pyrodinium bahamense var.

compressum from the Philippines. USA: Elsevier.

Azanza, R.V., Cruz, L.J., Carino, F.A., Blanca, A.G. & Butardo, V.M. (2009). Paralyticshellfish toxin concentration and cell density changes in Pyrodinium bahamense –

Noctiluca scintillans feeding experiments. Toxicon, 3(109)

Azanza, R.V., Dela Rosa, A., Sombrito, E.Z., Cruz, L., Siringan, F.P., McGlone, M.S.D., &

Duyanen, J. (2001). Harmful algal bloom (HAB) management lessons from

multidisciplinary research program in Manila Bay, Philippines. Philippines: DOST.

Badylak, S., Kelly, K., & Philips, E. (2004). A description of Pyrodinium bahamense

(Dinophyceae) from the Indian River Lagoon, Florida, USA. Phycologia, 43(6), 13-

17.

Bajaras, F., Relox Jr., J., & Fukuyo, Y. (2006). PSP in the Philippines: three decades of monitoring a disaster. Coastal Marine Science, 30(1), 104-106.

Busine, M.B., Cardenas, J., Khonghun, G., Pelobello, M.R., Raymundo, E., & Reyes, C. C.

(2002). The killer tide: The impacts and monitoring of red tide. Ekolohiya, 1(1), 1-8.

Carstensen, E.L., Miller, M.W., & Linke, C.A. (1974). Biological effects of ultrasound.

Journal of Physical Biological Sciences, 2, 173-192.

Cell counting and dye exclusion viability assays using haemocytometer. Tech Note (2004),

3(25), 1-2.

Chudleigh, T., & Thilaganathan, B. (2004). Obstetric ultrasound (3rd Ed.). Philadelphia,

USA: Elsevier Limited.

Emery, R.J., Papadaki, M., Freitas dos Santos, L.M., & Mantzavinos, D. (2005). Extent of

sonochemical degradation and change of toxicity of a pharmaceutical precursor

(triphenylphosphine oxide) in water as a function of treatment conditions.

Environment International, 31, 207-211.



24

Falconer, I. R. (1999). An overview of problems caused by toxic blue-green algae

(Cyanobacteria) in drinking and recreational water. Environmental toxicology, 14, 5 –

12.

Fernandez, D., & Ricafrente, M.V. (2010). DOST-PCAMRD supports the PhilHABs

program. The PCAMRD Waves, 1(23), 1-6.

Fleischer, A., Romero, R., Manning, F., Jeanty, P., & James Jr., A.E. (1991). The principles

and practice of ultrasonography in obstetrics and gynecology (4th Ed.). Connecticut,

USA: Prentice-Hall International Inc.

Food and Agriculture Organization of the United Nations. (2007). Installation and operation

of a modular bivalve theory. UK: Author.

Goldman, C.R., & Horne, A.J. (1983). Limnology. California: McGraw-Hill Inc.

Hallegraeff, G.M., Anderson, D.M., Cembella, A.D., & Enevoldsen, H.O. (1995). Manual on

harmful marine microalgae. Place de Fontenoy, Paris: UNESCO.

Karlson, B., Cusack, C., & Bresnan, E. (2010). Microscopic and molecular methods for

quantitative phytoplankton analysis. Place de Fontenoy, Paris: UNESCO.

Jaymalin, M. (1997, May 7). The moribund shellfish industry. The Philippine Star , pp. 1, 17.

Jinhui, W. (2005). The ecological engineering of HAB: Prevention, control, and mitigation of

harmful algal blooms. Electronic Journal of Biology, 1(2), 27-30.

Kinne, O. [Editor], Blaxter, J.H.S., Collier, A.W., Gunkel, W., Helleburst, J.A., & Segal, E.

(1970). Marine ecology, v. 1.Environmental Factors, Part 1. London: Wiley-

Interscience.

Lee, R.L. (1989). Phycology (2nd Ed.). New York: Cambridge University Press.

Lee, S. (2004). Ultrasonic cleaning baths. Retrieved from: http://www.impact

test.com/docs/SV050_055HB.pdf

Li, H., Huai, X., Cai, J, & Liang, S. (2009). Experimental research on antiscale and scale

removal by ultrasonic cavitation. Journal of Thermal Science, 18(1), 65-73.

Maclean, J.L. (1977). Observations on Pyrodinium bahamense plate, a toxic dinoflagellate,

in Papua New Guinea. Limnology and Oceanography. 22(2), 234-254.

Madison Area Technical College, Biotechnology Project. (2005). An overview of sonication.Wisconsin: MATC.

Newcombe, G. (2009). International guide manual for the management of toxic

cyanobacteria. London: GWRC.

Nybakken, J.W. (1982). Marine Biology, an Ecological Approach. New York: Harper &

Row.



25

O’Brien, W.D. (2007). Ultrasound-biophysics mechanisms. Progress in Biophysics and

Molecular Biology. Urbana, IL: Elsevier, 93, 212-255.

Olympus (2006).Ultrasonic transducers technical notes. USA: Author.

Oyib, D.H. (2009). Control mechanism of algal growth. Everything About Water ,11, 40-41.

Relox Jr., J., & Bajarias, F. (2003). Harmful algal blooms (HABs) in the Philippines.

Retrieved from: http://fol.fs.a.u-tokyo.ac.jp / /rtw/TOP/EXabst/019JuanRReloxJr.pdf

Ryding, S.O., & Rast, W. (1989). The control of eutrophication of lakes and reservoirs.

Cornforth, UK: Parthenon Publishing Group.

Sassi, J., Viitasalo, S., Rytkonen, J., & Leppakoski, E. (2005). Experiments with ultraviolet

light, ultrasound, and ozone technologies for onboard ballast water treatment.

Finland: Julkaisija-Utgivare.

Sherman, C.H., & Butler, J.L. (2007). Transducers and arrays for underwater sound. New

York: Springer.

Song, W., Teshiba, T., Rein, K., O’shea, K. E. (2005). Ultrasonically induced degradation

and detoxification of Microcystin-LR (cyanobacterial toxin). Environmental science

& technology, 39 (16), 6300 – 6305.

Using ultrasound to control toxic algal blooms. (2010). Retrieved from

http://www.physorg.com/news197715172.html

Usup, G., Kulis, D., & Anderson, D. (1994). Growth and toxin production of the toxic

dinoflagellate Pyrodinium bahamense var. compressum in laboratory cultures.

Natural Toxins, 2, 254-262.

Van Iersel, M.M. (2008). Sensible sonochemistry. Eindhoven: Eindhoven University.

Villao, R.S. (1988). The red tide menace. Diliman Review, 36 (5), 44-45.



26

APPENDICES

Appendix A. Summary of Materials and Methods

Figure 5.1. Process Flowchart of the Research Study

Procurement of stock culture

containing P. bahamense var.

compressum (A)

Preparation of culture flasks

containing P. bahamense var.

compressum (n = 24) (B)

Labeling, Grouping, and

Randomization of Solutions (C)

Acquisition of an ultrasonic

sonicator (D

Acquisition of Sedgewick-Rafter slide (E)

Acquisition of other lab equipment (F)

Setting up of Ultrasonic Cleaner (G)

Exposure of Algae to Ultrasound (Algae vs.

Time of Exposure) (I)

Final Cell Counting (2 trials) (J)

Initial Cell Counting (2 trials) (H)

Statistical Tests (ANOVA and T-Test) (L)

Graphing and Tabulation of

Collected Data (K)



27

Appendix B. Formulas, Tables, and Graphs needed for Data and Analysis

Table 5.2.1. Table for Analysis of Variance (ANOVA)

Source of

Variation

Sum of

Squares

Degrees of

Freedom

Mean

Square

Fcalc F tab or

Fcrit Treatments SSTr DFTr MSTr

Error SSE DFE MSE

TOTAL SST DFT

∑ ∑

n = Total number of samples nk = Number of samples per treatment

k = Number of Treatments Tk = Treatment Totals

If Fcalc > Ftab , reject null hypothesis. If Fcalc < Ftab, accept null hypothesis.

Table 5.2.2. Table Comparing Initial and Final Cell Density for each Treatment

TREATMENT NO: 1

Flask # Initial Cell Density Final Cell Density Change In Cell Density (D)

21TRIAL 1

TRIAL 2

4TRIAL 1

TRIAL 2

Table 5.2.3. Table for Correlated T-Test

TREATMENT NO: 1

Flask # D D2

21TRIAL 1

TRIAL 2

4TRIAL 1

TRIAL 2

SUMMATION ()

Equations for Correlated T-Test:

∑ ∑ ∑ √ ∑

∑



28

n = number of sample pairs = average change in cell density

D = change in cell density ∑ = sum of squares of the difference

= mean difference

If t > tabulated value for t, accept alternative hypothesis, and reject null hypothesis. If t <

tabulated value for t, accept null hypothesis, and reject alternative hypothesis.

Table 5.2.4. Initial, Final, and Change of Cell Densities of Pyrodinium bahamense var.

compressum cultures in Concentration of Algae vs. Time of Exposure

Treatment C T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11

Flask #

Initial Trial 1

Trial 2

Change Trial 1

Trial 2

Final Trial 1

Trial 2

Legend: All treatments are exposed with 1 MHz ultrasound

C – no exposure to ultrasound T-4 – 2.5 hr of exposure T-8 – 4.5 hr of exposure

T-1 – 1 hr of exposure T-5 – 3 hr of exposure T-9 – 5 hr of exposure

T-2 – 1.5 hr of exposure T-6 - 3.5 hr of exposure T-10 – 5.5 hr of exposure

T-3 – 2 hr of exposure T-7 – 4 hr of exposure T-11- 6 hr of exposure



29

Appendix C. Risk Assessment

Table 5.3. Risk Assessment Table

Substance/Device/Organism Risks/Dangerous Effects Safety Procedures

Pyrodinium bahamense var.

compressum

*NOTE: Medical assistance

must be present during

experiment of this kind of

harmful algae.

- Contains saxitoxin (STX), a

dangerous toxin.

- STX can cause gastrointestinal,

respiratory, and neural symptoms.

Symptoms will start by vomiting and

paralysis. Paralysis will be

succeeded by dysphagia, and then

death.

- Fatalities were usually a result of

respiratory failure.

There is no specific antidote for PSP,

and the manner

of treatment is purely symptomatic.

- Before and after handling this

algae, hands must be washed withsoap and water

- Researchers must wear a lab gown,

elbow-length puncture-resistant

gloves, and boots.

- ONLY use mechanical pipetting

for algal transfer

- No eating, drinking, or applying of

cosmetic products during work time.

- Excess algae must be disposed in aleak-proof reagent bottle.

- All other living things unrelated to

the study are prohibited inside the

lab that contains the algae.

Seawater - Slightly hazardous in case of

skin/eye contact (irritant), ingestion,

or inhalation.

- May affect behavior (muscle

spasicity/contraction, somnolence),

sense organs, metabolism, and

cardiovascular system. Continuedexposure may produce dehydration,

internal organ congestion, and coma.

Inhalation: Material is irritating to

mucous membranes and upper

respiratory tract.

- When heated to decomposition it

emits toxic fumes.

- Electrolysis of sodium chloride in

presence of nitrogenous compounds

to produce chlorine may lead to

formation of explosive nitrogen

trichloride. Potentially explosivereaction with dichloromaleic

anhydride + urea.

- Hygroscopic. Reacts with most

nonnoble metals such as iron or

steel, building materials (such as

cement) Sodium chloride is

- Precautions: Keep locked up.. Do

not ingest. Do not breathe dust.

Avoid contact with eyes. Wear

suitable protective clothing. If

ingested, seek medical advice

immediately and show the container

or the label. Keep away from

incompatibles such as oxidizingagents, acids.

- Eye Contact: Check for and

remove any contact lenses. In case

of contact, immediately flush eyes

with plenty of water for at least 15

minutes. Cold water may be used.

Seek medical attention

- Skin Contact: Wash with soap and

water. Cover the irritated skin with

an emollient. Get medical attention

if irritation develops. Cold water

may be used.

- Inhalation: If inhaled, remove to

fresh air. If not breathing, give

artificial respiration. If breathing is

difficult, give oxygen. Get medical

attention if symptoms appear.



30

rapidly attacked by bromine

trifluoride. Violent reaction with

lithium.

- Mutagenic for mammalian somatic

cells. Lowest Published Lethal Dose

(LDL) [Man] - Route: Oral; Dose:

1000 mg/kg

-Ingestion: Do NOT induce

vomiting unless directed to do so by

medical personnel. Never give

anything by mouth to an

unconscious person. Loosen tight

clothing such as a collar, tie, belt or

waistband. Get medical attention if

symptoms appear.

- Personal Protection: Splash

goggles. Lab coat. Dust respirator.

Be sure to use an approved/certified

respirator or equivalent. Gloves.

- Accidental Small Spill: Use

appropriate tools to put the spilled

solid in a convenient waste disposal

container. Finish cleaning by

spreading water on the contaminated

surface and dispose of according to

local and regional authorityrequirements.

- Accidental Large Spill: Use a

shovel to put the material into a

convenient waste disposal container.

Finish cleaning by spreading water

on the contaminated surface and

allow evacuating through the

sanitary system.

- Waste Disposal: Waste must be

disposed of in accordance with

federal, state and localenvironmental control regulations.

Lugol’s Iodine - Hazardous in case of ingestion.

Slightly hazardous in case of skin

contact ( irritant, permeator), of eye

contact (irritant).

- Mutagenic for mammalian somatic

cells. Classified Reproductive

system toxin for females. The

substance is toxic to thyroid. The

substance may be toxic to blood,

kidneys, liver, skin, eyes. Repeated

or prolonged exposure to thesubstance can produce target organs

damage.

- Potassium iodide (KI) + Fluorine

Perchlorate (FClO4) will explode on

contact.

- Eye Contact: Check for and

remove any contact lenses. In case

of contact, immediately flush eyes

with plenty of water for at least 15

minutes. Cold water may be used.

Get medical attention.

- Skin Contact: Wash with soap and

water. Cover the irritated skin with

an emollient. Get medical attention

if irritation develops. Cold water

may be used.

- Ingestion: Do NOT induce

vomiting unless directed to do so by

medical personnel. Never give

anything by mouth to an

unconscious person. Loosen tight

clothing such as a collar, tie, belt or



31

- Slightly reactive to reactive with

oxidizing agents, reducing agents,

organic materials, metals, acids.

- Acute Potential Health Effects:

Skin: Causes skin irritation. It can

cause brown stains on the skin. It

can be absorbed through the skin.

Eyes: Eye contact with liquid causes

irritation. Iodine vapors may cause

eye irritation. Eye contact with an

excessive amount of iodine vapor

may also cause blepharitis.

Excessive inhalation of iodine

vapors may cause respiratory tract,

nasal, and mucous membrane

irritation. Symptoms may include

coughing, tightness in the chest,

rhinitis, dyspnea/respiratory distress,

coughing, sneezing, pulmonary

edema, chemical pneumonitis,edema of the larynx and bronchi,

pharyngitis, swelling of the parotid

gland, and cachexia. High exposure

may lead to lung disease and may

also affect behavior/central nervous

system (delirium, hallucination,

depression, seizure)

waistband. Get medical attention if

symptoms appear.

- Inhalation: If inhaled, remove to

fresh air. If not breathing, give

artificial respiration. If breathing is

difficult, give oxygen. Get medical

attention.

- Hygienic Practices: Avoid contact

with eyes, skin and clothing. Wash

hands after direct contact. Do not

wear product-contaminated clothing

for prolonged periods.

- Engineering Controls: Provide

exhaust ventilation or other

engineering controls to keep the

airborne concentrations of vapors

below their respective threshold

limit value.

- Personal Protective Equipment:

Splash goggles. Lab coat. Gloves.

- Spill Procedures: Dilute with water

and mop up, or absorb with an inert

dry material and place in an

appropriate waste disposal container.

- Waste Disposal: Dispose of in

accordance with all applicable

federal, state, and local regulations.

Sonicator - Safety concerns relating to

ultrasound technology are possible

noise from the transducer, yet

unknown effects upon humans

affected by the exposure to

ultrasound.

-Heat is generated in the transducer

if the cooling system fails.

- Sonicators are usually constructed

of steel, titanium, aluminium or

ceramic material.

- Sonicators develops noise (if set at

high frequencies) that can irritate

humans and animals (>1 MHz)

- Do not install the sonicator in a

hot/humid area. Place it in an area

with proper ventilation

- Check the connection cable for

damage due to overheating/moisture

- If the generator main fuse blows,

check the generator for a short

circuit, the on/off switch,

connection of the supply

transformer, printed circuits for

blown fuses

- Clean out dirt inside and outside of

the sonicator.

- Wipe/dry ultrasonic cleaner after

use.



32

Appendix D. Task List

Table 5.4. Task List of the Methods in the Research Study

ACTIVITYCODE

ACTIVITYDESCRIPTION

OBSERVABLEINDICATORS

PRECEEDINGACTIVITY

ESTIMATEDDURATION

A Procurement of stock culture

containing

Pyrodinium

bahamense var.

compressum

Stock cultureobtained from MSI

B 1

B Preparation of

culture flasks

containing

Pyrodinium

bahamense var.

compressum

Twenty-four 125 mL

Erlenmeyer flasks

with 100 mL AlgalSolutions

C 1

C Labelling,

Grouping, and

Randomization of Solutions

24 flasks are labeled

acc. to where it

belongsFlasks are

randomized using

CRD

G 1

D Acquisition of an

ultrasonic sonicator

Ultrasonic cleaner

obtained from I-MAT Pro Company

c/o Mr. Publico

G 7

E Acquisition of an

Sedgewick-Rafter

silde

Sedgewick-Rafter

slide obtained from

MSI

G 1

F Acquisition of

other lab equipment(beakers, pipette,

droppers,

microscopes,autoclave, etc.)

Microscope, pipette,

autoclave, stirringrod, regeant bottles,

seawater, Eppendorf

tubes, Lugol’sIodine, etc.

G 1

G Setting Up of Ultrasonic Cleaner

Ultrasonic Cleaner isalready switched on

with 2 flasks in a

treatment (Repeated

11 times, one foreach treatment)

H 1

H Initial CellCounting and

Cell Densities of each flask are

I 5



33

Computation of

Initial Cell Density

obtained. 2 trials

each

I Exposure of Algae

to Ultrasound

(Algae vs. Time of

Exposure orExperiment A)

All 11 treatments are

done (All flasks

except control are

exposed toultrasound)

J 5

J Final Cell Counting All 1 mL samples of

each flask are

counted (2 trials

each flask)

K 5

K Graphing andTabulation of

Collected Data

Graphs and Tablessaved in Excel

L 1

L Statistical Tests ANOVA, andCorrelated T-Test

performed with 0.05level of significance

_ 1



34

Appendix E. Materials Sourcing and Budgeting

Table 5.5.1. Table of Materials needed for Experiment and their Costs

Quantity – MaterialNeeded

Source Address Contact Payment

1 - Autoclave15 - Beaker

1 - Microscope

2 – Single ChannelPipette

24 – 125 mL Erlenmeyer

FlasksBolinao Seawater

Cheesecloth

Cotton

Alcohol Lamp

CHEMICALS:

F/2 medium

Lugol’s Iodine

MarineScience

Institute (MSI)

Velasquez Street, UPDiliman, Quezon City

EsrelitaFlores

921-5967

922-3957

Free of Charge

1 – Ultrasonic Cleaner I-MAT ProCompany

Kentwood Heights,Mariposa Street, Brgy.

Crame, Quezon City

RamonPublico

Free of Charge

1 - Stock culture of

Pyrodinium bahamense

var. compressum

Marine

Science

Institute

Velasquez St., UP

Diliman, Quezon City

Esrelita

Flores

921-5967

922-3957

Free of

Charge

4 – Sedgewick RafterCounting Slides

MarineScience

Institute

Velasquez St., UPDiliman, Quezon City

EsrelitaFlores

921-5967

922-3957

Free of Charge

Table 5.5.2. Table of Transportation and Electricity Consumption

Date of Work Consumption Duration or Price

February 24, 2011

Justin’s Car – Round Trip:

PSHS to MSI

15 minutes

Water Filterer (Useselectricity)

30 minutes

Autoclave (Uses Electricity) 1 hour

February 25, 2011

Commuted: Round Trip:

PSHS to MSI

Taxi – Php 80.00

FX to Agham Road (twice) –

Php 20.00



35

Jeep (UP Ikot) – Php 7.00

Jeep (UP to Agham Road) –

Php 10.00

Total: Php 117.00

Microscope Viewing (Uses

Electricity)

2 hours

February 28, 2011 Justin’s Car – Round Trip:PSHS to MSI

15 minutes

Microscope Viewing 2 hours

March 1, 2011 Justin’s Car PSHS to I-MAT Pro

Company – 1 hourI-MAT Pro Company to MSI

(twice) – 30 minutes*2 = 1

hour

MSI to I-MAT Pro Company

– 1 hour

MSI to PSHS = 10 mintuesTotal = 3 hrs. 10 minutes

Microscope Viewing 2 hours

Use of Computer (Letter of

Documentation Editing)

30 minutes

March 4, 2011 Justin’s Car – Round Trip:

PSHS to MSI

15 minutes

Ultrasonic Cleaner 1 hour

Daily Lamp (for Algae Control) 12 hours each day



Appendix F. Gantt Charts

Figure 5.6. Gantt Chart with Expected Dates of Work

*For legends, see Appendix D (Task List)

Annex E. Network Chart

Figure 5.7. Network Chart of the Research Study

A

B

C

D

E

F

G

H

I

J

K

L

23-Feb 24-Feb 25-Feb 26-Feb 27-Feb 28-Feb 1-Mar 2-Mar 3-Mar 4-Mar 5-Mar 6-Mar 7-Mar 8-Mar 9-Mar 10-Mar 11-Mar 12-Mar 13-Mar 14-Mar 15-Mar

DATE (DAYS)

Feb 23 - START, March 15 - END

1

6

52

3

11

13

7

4

9

12

8

C = 1 d

B = 1 d

A = 1 d E = 1 d

10

D = 7 d

G = 1 d

H = 5 d

I = 5 d

K = 1 d

L = 1 d

F = 1 d

J = 5 d

Final Proposal Rb09

Documents

Transcript of Final Proposal Rb09