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CHAPTER IV

Research Methodology

4.1 Research Design

The design of this research is true experimental post test only control

group design in order to prove Black cumin (Nigella sativa L) has potential as

effective natural insecticide (quick knockdown).

Table 4.1 Groups and Type of Treatment

No Groups of Treatment Type of Treatment

1 Negative control Sprayingwith1% acetonecomponent(without
the extract ofblack cumin(Nigella sativa))

2 Positive control Sprayingwith 0,28% malathion (without the

extract ofblack cumin(Nigella sativa))

3 Treatment 1 Sprayingwithextracts ofblack cumin(Nigella

4 Treatment 2
5 Treatment 3
6 Treatment 4
sativa) with a concentration of10%
Sprayingwithextracts ofblack cumin(Nigella
sativa) with a concentration of25%
Sprayingwithextracts ofblack cumin(Nigella
sativa) with a concentration of50%
Sprayingwithextracts ofblack cumin(Nigella
sativa) with a concentration of75%

4.2 Population and Sample

4.2.1 Population
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Population of this research is adult mosquito.

4.2.2 Sample

The sample of this research is adult mosquito from Aedes agypti

that fulfilled inclusion criteria. The criteria is alive and move actively. For

the exclusion criteria is adult mosquitoes died before the study.

Total mosquitoes of this study is 120 on each repetition. For

mosquitoes preparation, we will collaborate with staff from Department of

Parasitology, University of Brawijaya Malang to provide adult Aedes sp

mosquito strain. For identification purpose our study will be supervised by

major advisor (dr. Muhammad Luthfi Al Manfaluthi, DTM&H, MCTM).

This study will use four different active dose concentration (15%, 25%,

50% and 75%), positive control malation (0,28%), negative control

(aquades).

Repetition formula according to Tjokronegoro 2011:

P(n-1) ≥ 16

P (n-1) ≥ 16

6 (n-1) ≥ 16

6n-6 ≥ 16

6n ≥ 22

n≥3,6
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4.3 Time and Place the research

The research will be conduct in laboratory biomedic,Faculty of

Medicine, University of Muhammadiyah Malang in May 2014.

4.4 Variable Research

4.4.1 Independent variable

Independent variable is concentration Black cumin (Nigella sativa L) (in

15%, 25%, 50%, and 75%).

4.4.2 Dependent Variable

Dependent Variable is knockdown time

4.5 Methods

Our experiment will be divided in three steps:

1) Step 1 (mosquito and chamber preparation)

For mosquitoes preparation, we will collaborate with staff from

Department of Parasitology, University of Brawijaya Malang to provide

adult Aedes aegypti sp mosquito strain. A total mosquitos will be needed

for this experiment is 520. 20 mosquitoes for each chamber multiply with

6 (different concentration from 10%, 25%, 50%, 75%, 0.28% malathion

and 1% aceton) is 120 mosquitoes. According to our repetition formula,

we will perform 4 times repetition so 120 multiply with 4 are 480. In order

to prevent drop out phenomenon (in this case mosquitoes die within

acclimatisation phase) we prepare 10% more stock to overcome if this

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condition happening. 10% from 480 is 48 so we add 480 + 48 = 520

mosquitoes in this experiment.

For Chamber preparation our major advisor will provide 6

artificial cage 25x25x25 cm3 with one big hole 15 cm in diameter covered

by 50 cm in length circular net. That net will be used to put in both the

mosquitos and the sprayer contain the active extract also for the glucose

solution.

Pictures 4.1 Mosquito chambers

2) Step 2 (extractpreparation)

For extract preparation, first we will provide fresh Black cumin seeds

for 100 grams as the raw material, second we prepare extraction and

evaporation equipment such as: measuring cup, stove, fabric filters, scales,

aluminum foil paper, abeaker glass,blender, 1L erlenmayer tube, oven and

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evaporation set (container tube, spiral cooler, rotate-extraction tube, water

bath, vacuum, static clem, plastic tube, aquades container, extraction tube)

Pictures 4.2 Erlenmeyer tube and measuring cup

Procedure :

The part from Black cumin plant that will be used in this

experiment is seedss only. Black cumin seedss will be cleaned with clean

water before getting slice in small inch and proceed to be dried under sun-

ray for 3 hours. Next, the seedss will be put inside the microwave until

perfect dry with temperate 70 degree Celsius. Dry Black cumin seedss will

be blended until be powder form and separated every 100 mg. Black

cumin powder will be sunk with ethanol for a night in 1 L Erlenmayer

flask. The next day this extract will be separated from ethanol solvent

with evaporation procedures.

Evaporator machine will be put in 30 - 40 degree in order to make

fixation to the tube. 1 night Black cumin powder with ethanol will be
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moved to separated chamber linked with cold spiral and plastic vacuum

tube connected with water pump filled with cold water. Water pump will

be placed in aquades, and connected with electrical source until aquades

filled cold spiral tube. 1 set evaporation will be placed in half of separated

extraction sinked in water bath. Vacum and water bath will be connected to

electrical source and the temperature will be increased until 70 degree

Celcius (upper limit ethanol evaporation). Let the machine flow

approximately 4 hours then continued with heating up until 50-60 degree

Celcius for 1-2 days. The last process evaporation will make 100%

concentration of Black cumin seedss. In order to prevent the degeneration

process or make the extract durability, we can safe under refrigerate

maximum for 30 days before discarding. In our experiment, Black cumin

extract will be provided by Staff from Departement of Biochemistry

Faculty of Medicine, University of Brawijaya Malang.

To get the desired concentration we will conduct dilution from

100% pure extract with aquadest by 1% aseton. Aceton is used due to its

ability to solve non-polar liquid like aquades. Formula for dilution is

mention below:

M1 x V1 = M2 x V2

Description:

M1: concentration of the stock solution of the magnitude of

the 100%

M2: concentration of the desired solution

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V1: the volume of stock solution should be diluted

V2: the volume of a solution of the desired treatment

The final Volume of solution treatment required for each concentration is

3.5 ml. so, after taken from the stock solution is then added to the aquadest sterile

until it reaches avolume of 3.5 ml

There will be 4 kind experiments with different doses. The doses to

be used are 10%, 25%, 50% and 75%. To obtain the expected volume of

3.5 ml with a concentration of 10%, it can be making as much as 4 ml

stock solution using the calculation:

100% x V1 = 10% x 4 ml

V1 = 0,4 ml

V1 is the volume of black cumin (Nigella sativa) extract, dilution

is done by adding a mixture of aquades plus 1% acetone as much as 3.6

ml.

To obtain the expected volume of 3.5 ml with a concentration of

25%, it can be making as much as 4 ml stock solution using the

calculation:
100% x V1 = 25% x 4 ml

V1 = 1 ml

V1 is the volume of black cumin (Nigella sativa) extract, dilution

is done by adding a mixture of aquades plus 1% acetone as much as 3 ml.

To obtain the expected volume of 3.5 ml with a concentration of

50%, it can be making as much as 4 ml stock solution using the

calculation:
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100% x V1 = 50% x 4 ml

V1 = 2 ml

V1 is the volume of black cumin (Nigella sativa) extract, dilution

is done by adding a mixture of aquades plus 1% acetone as much as 2 ml.

To obtain the expected volume of 3.5 ml with a concentration of

75%, it can be making as much as 4 ml stock solution using the

calculation:

100% x V1 = 75% x 4 ml

V1 = 3 ml

V1 is the volume of black cumin (Nigella sativa) extract, dilution

is done by adding a mixture of aquades plus 1% acetone as much as 1 ml.

3) Step 3 (knockdown experiment)

After 3 days adaptation in mosquitos chamber with additional glucose for

mosquito’s food supply, we will perform knockdown test.

First, we will withdrawal the glucose (food supply) and make spraying for

the cage both direct to the mosquitoes and the wall of inner cage. We will spray

with different concentration from the lowest 10%, 25%, 50%, 75% also 2
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additional concentrations for both positive control 0.28% malathion and negative

control 1% acetone as solvent from our extract.

Second, we will record for dying mosquito and try to push with stick to

provoke the movement. Any kind of movement means the mosquito still alive, so

we will not put under knockdown status. we will evaluate from the time of

spraying (minute-0) until an hour (minute-60) every 5 minutes. (0, 5, 10, 15, 20,

25, 30, 35, 40, 45, 50, 55, 60, 1440)

Third, after 60 minutes, we will seeds the cage with new glucose solution

for mosquitoes food in case some of them still alive to maintain the survival until

the next 23 hours.

Fourth, for the next day will re-evaluate the last record to make sure

whether the mosquitoes die due to our extract or they just get wet or dying due to

inability to fly.

Picture 4.3 Timer and Spray

4.6 Operational Definition

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Black cumin extract is evaporation product from dry extraction Black cumin

seedss with ethanol, which have characteristic oil based form, un-dissolve to

water and contain 100% concentrate.

Positif control is comparison product that more valid in evidence and already

being standard in daily practice. In our experiment we will use 0.28 malathion

compare to our extract with different concentration from 10%, 25%, 50% and

75%. For negative control, we will use 1% acetone, as the solvent of our extract

that contain no insecticide activity.

Adult Aedes aegypti sp mosquitoes will be gained from staff in Department of

Parasitology, Faculty of Brawijaya University with characteristic:

- Adult mosquitoes Aedes aegypti with alive and fly actively

Knockdown time is amount of time needed to make mosquito down after the

treatment. In our experiment, we will measure after spraying with Black cumin

extract from 0-60 minutes with every 5 minutes interval.

Mosquito Chamber is a special box cage with 25 x 25 x 25 cm3 with one big

hole 15 cm in diameter covered by 50 cm in length circular net. That net will be

used to put in both the mosquitos and the sprayer contain the active extract also

for the glucose solution.

Glucose solution 10% is a mixture of glucose and water in order to provide food

supply within acclimatisation phase and during 60 minutes until 1440 minutes

(24 hours) after spraying.

4.7 Observation
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Observation will be conducted in minutes 0, 5, 10, 15, 20, 25, 30, 35, 40,

45, 50, 55, 60 and 1440. Falling mosquitoes will be manipulate by stick to make

sure whether it is died already or just dying or wet due to spraying method. The

result from first until fourth repetition will be summarise before data being

analysis.

Analysis will be used software SPSS version 22 for analysis of variance

(ANOVA) and post hoc test. This experiment use 6 different group with

independent type of data. Here is the step of analysis:

1. Perform descriptive statistic or frequency to explore mean, median, mode,

quartile, standard deviation related with knockdown profile of Black cumin

extract.

2. Check the requirement for parametric test (ANOVA test)

1. Type of independent variables should be numeric data

2. Perform marginal homogenity test in explore function, in this study we

will use kolmogorov smirnov instead shappiro-wilk test due to our sample

size is larger than 50. The result of this test will inform the data

distribution whether normal (p-value more than 0.05) or the opposite.

3. Perform Leuvene test in order to check the data varians, if P-value more

than 0.05 means the variant does not normal.

4. In case the requirement of parametric test does not fit, we will perform

non-parametric test according set data. we will utilise kruskall-walls test or

friedman test as alternative statistical test.

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3. After ANOVA test we will proceed to post hoc test to explore possibilities

within sub-group. we will use Duncan, LSD, Bonferonni test according to the

result in the near future.

4.9 Chart of Research

Insert the mosquitoes, aedes

agypti each 20 tails

Chamber 1 Chamber 2 Chamber 3 Chamber 4 Chamber 5 Chamber 6

Control + Control – Black Black Black Black
(acheton (malathion cumin cumin cumin cumin
0,28%) extract extract extract extract
1%)
15% 25% 50% 75%

Observed once every 5 minutes for up to 60 minutes

Records the result obtained every 5 minutes

Evaluation after 24 hours

Repeated 3 times

Data Analysis
4.10 Data Analysis

Data obtained in this study in the analysis of using One Way ANOVA and

Post Hoc Turkey HSD of processing using the SPSS version 22.
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1. One Way ANOVA

Used to prove the existence of a meaningful difference between the

control and treatment. ANOVA test results there was significant if <

0.01.
2. Post Hoct Turkey HSD
The test is a continuation of the ANOVA test, used to determine a

meaningful difference between the respective research groups.

Time-Schedulle

April April May

April 2014
Activity 2014 2014 2014
(early)
(mid) (late) (early)
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Topic Selection

Outline Proposal

Final Proposal

Proposal defense

Data Collection

Outline Thesis

Final Thesis

Thesis defense