CHAPTER III

Methodology

This chapter will present the kind of research and the

research design the researchers used in the study. Also included

here are the materials and methods used.

Kind of Research

This study is an experimental research. The Annona muricata

leaf extract was tested on Periplaneta americana through

spraying to determine its insecticidal property. An

experimental research is a kind of research wherein the

researchers try to get results from the dependent variables

after subjecting it to different treatments. In this study, the

Annona muricata leaf extract was the independent variable. It

was manipulated by the researchers as they subjected the extract

into different concentrations. The dependent variable of this

experimental research was the mortality rate of the cockroaches.

Research Design

This research followed a Time Series Design. Quantitative

observations followed evenly spaced intervals and measured

successively from the different extract concentrations. The

cockroach cages were sprayed by different concentrations and

were observed in every 30 minutes for a course of 2 hours. There

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were one hundred twenty (120) individual cages for the

cockroaches. Thirty (30) cockroaches were exposed on each

treatments.

There were four (4) treatments in this experiment. The

Annona muricata leaf extract were diluted into 1 g of leaf

extract: 1 mL of distilled water and 1g of leaf extract: 3 mL of

distilled water concentration ratios. Two control treatments

were also prepared. A commercially available multi-insect killer

was the positive control, while the distilled water was the

negative control.

Figure 3.1. Design for arrangement per treatment on each cage of

Periplaneta americana

A21 A4 A6 A12 A24 A16 A15 A27 A17 A9

A26 A7 A30 A25 A11 A18 A10 A3 A2 A29

A8 A1 A23 A13 A19 A28 A5 A14 A22 A20

B2 B24 B13 B9 B5 B26 B7 B27 B22 B16

B28 B12 B11 B10 B1 B8 B4 B20 B19 B15

B17 B21 B23 B30 B18 B3 B14 B6 B29 B25

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 C7 C24 C9 C14 C26 C22 C18 C5 C12 C1

C4 C15 C13 C29 C19 C16 C8 C11 C17 C20

C27 C25 C30 C23 C21 C2 C6 C28 C3 C10

D14 D12 D3 D1 D2 D27 D5 D28 D9 D6

D11 D21 D10 D4 D24 D30 D15 D20 D26 D18

D23 D29 D19 D7 D25 D8 D13 D17 D22 D16

Legend:

A Treatment sprayed with 1:1 concentration ratio

of Annona muricata leaf extract
B Treatment sprayed with 1:3 concentration ratio
of Annona muricata leaf extract
C Treatment sprayed with Commercially available
multi-insect killer
D Treatment sprayed with Distilled water

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Figure 3.2

Flow Chart of the Study

Construction of Cages

Collection of Annona
muricata leaves Purchase of
Periplaneta americana

Preparation of Annona
muricata leaves Sorting, and Selection of
(Drying and Grinding) Periplaneta americana

Extraction by Maceration of
Acclimatization
Annona muricata leaves

Preparation of Annona
muricata leaf extract
concentrations
(1:1 and 1:3 concentration
ratios) and the Negative
and Positive Control

Spraying of Annona muricata leaf

extract on
Periplaneta americana

Observation and Recording of

Results

Statistical Treatment

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Materials and Methods of the Study

Construction of Cages

One hundred twenty (120) rectangular cages were

constructed, made up of mesh wires with 3 mm size holes having

the dimensions of 6cm x 5cm x 10cm.

Purchase of Periplaneta americana

Two hundred (200) samples of Periplaneta americana were

bought from Aranque Market, Manila. The cockroaches were placed

in a secured sack.

Sorting and Selection of Periplaneta americana

Purchased cockroaches were sorted out to be homogenized.

The cockroaches underwent standard deviation. The length of each

cockroach was measured from the tip of the head up to the end

tip of the abdomen. The range calculated was 27 mm up to 33 cm.

Cockroaches that measured within the range were selected. Sorted

out cockroaches were placed on individual cages.

Acclimatization of Periplaneta americana

A tent was built to provide protection from rain and other

external factors. The tent was made up of tarpaulin canvass

framed with wood and shaped as a tent occupying a total land

area of three (3) square meters. It was assembled behind the

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Maceda Building. The sorted cockroaches were left there for two

days before the actual spraying of the insecticide to let them

acclimatize or adapt on the environment. They were provided with

leftover steamed rice to serve as their source of nourishment. A

wet cotton balls was also placed in the cage to serve as the

cockroaches hydration. It was replenished everyday. After two

days, weak or dead cockroaches were removed and viable

cockroaches were selected.

Collection of Annona muricata Leaves

Three (3) kilograms of fresh Annona muricata leaves were

collected at Barangay Maharlika, San Jose del Monte, Bulacan,

Philippines.

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Preparation of Annona muricata (Drying and Grinding)

Three (3) kilograms of the collected Annona muricata leaves

were washed and sun dried for 2 days. It was ground into powder

using a blender. Using an analytical balance, the resulting

weight of the grounded powder was 324.8 g.

Extraction by Maceration of Annona muricata Leaves

The powdered Annona muricata leaves were macerated using

ethanol as the solvent in 1:4 ratio. Three hundred grams (300 g)

of the powder were soaked in 1200 mL of 95% ethanol for 24 hours

at room temperature. Using a cheesecloth, the mixture were

filtered. The filtrate was filtered again using a funnel and

filter paper. Eight hundred mL ethanolic extract was made. The

solvent was removed using a rotary evaporator at Adamson

University Chemical Laboratory. The crude extract from the

Annona muricata leaves was stored in an amber bottle. It was

weighed and measured to have 12. 369 g.

Preparation of Annona muricata Leaf Extract Concentrations

There were four treatments prepared. Two concentrations

were derived from the extract, 1:1 and 1:3 concentration ratio.

The 1:1 concentration ratio was made by diluting 9 g of the leaf

extract to 9 mL of distilled water. While, the 1:3 concentration

ratio was made by diluting 3 g of the leaf extract to 9 mL of

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distilled water. A positive and negative control was also

prepared. Commercially used multi-insect killer served as the

positive control. Distilled water served as the negative

control. Nine (9) mL of the prepared concentrations and 9 mL of

the control treatments were transferred into different 60 mL

spray bottles with fine nozzles. Each was labeled accordingly.

Spraying of Annona muricata Extract on Periplaneta americana

Thirty (30) cages were sprayed with 9 mL of each

concentration. The insecticide was sprayed on the cages 8

oclock in the morning. The same procedure was applied for the

negative and positive control treatment. The spray bottle was 10

cm away from the cages.

Observation and Recording of Results

Two (2) hours were allotted for the observation of the

activity of the cockroaches. Using a timer, observations were

recorded every 30 minutes. The mortality of the cockroaches was

observed.

Statistical Analysis of the Gathered Data

The statistical tools used in this study were the Kaplan -

Meir Survival Curve and Log - Rank Test. The Kaplan - Meir

Survival Curve was used to compare the mortality of the

cockroaches on the different cages assigned for each of the

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concentrations. Log Rank Test was used to find out whether

there was a significant difference among the mortality of

cockroaches in terms of number killed and to be killed when they

are exposed to concentrations of Annona muricata leaf extracts.

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