Preparation of Extracts

Three kinds of leaf sample and three essential oils of cajuputi were named CA, CB, CC, CD, CE, and CF
in this report for convenience. CA was collected in a residential area at Palembang (Indonesia) in October
1999. CB was collected at the roadside of a mountain 15km from Yogyakarta (Indonesia) in July 2000.
CC was collected in a cajuputi essential oil factory in Sukun in the suburbs of Ponorogo (Indonesia) in
July 2000. CD was an essential oil obtained by hot water distillation from a tree growing at the University
of Gadja Mada. CE was the product of the above factory in Sukun and was obtained by steam distillation.
CF was a commercial product in Indonesia. CD and CF were provided by JIFPRO (Japan International
Forestry Promotion and Cooperation Center). In this experiment, all leaf samples (GA–GC and CA–CC)
and the leaves distilled for essential oil samples were collected from trees that were 3– 4 years old.

Essential oils

The essential oils of GA–GC and CA–CC were prepared by hot water distillation methods standardized
by the Association of Official Agricultural Chemistries.4 Fresh leaves were cut into pieces of less than 1
1cm, and 150- to 200-g samples were boiled with 1000 ml of deionized water for 16h.

Ethanol extraction

The fresh leaves of CB and CC (20–30g) were placed in ethanol (60ml). The capped samples were then
microwaved for 25s and were left to stand at room temperature for 3 days. After that the leaves were
filtered out, and the ethanol solution was concentrated to obtain ethanol extractives.

GC-MS analysis

Oil components were identified by gas chromatography– mass spectrometry (GC-MS) analysis. The GC
was equipped with a TC-FFAP column (30m 0.25mm) that was held at 60°C for 30 min and then heated
from 60°C to 210°C at 2°C/min using helium as the carrier gas. Mass spectra were obtained at 70eV.
Peaks were confirmed by library searches and comparisons with authentic samples.

Termiticidal Activity

In the bioassay for termiticidal activity, cut filter papers (diameter 25mm) were placed in each hole of a
six-hole multidish (3 rows 2 lines, hole diameter 25mm). Essential oils were diluted to 10.0% to 0.1% with
methanol, and specimens of some high-quantity compounds in essential oils were diluted with methanol
to the concentration at which they existed in the leaves. Samples of 50µl were placed on the filter paper in
each hole of one line. In the bioassay for contact termiticidal activity, six termites were put on one line on
which a sample was soaked on a filter paper. In the bioassay for noncontact termiticidal activity, six
termites were put on the other line, which did not contain a sample. The six-hole multidishes were closed
tightly and kept at 27°C in an incubator. The numbers of living termites were counted each day.

Repellent Activity

Filter paper was soaked with 10µl of a sample, and six termites were placed in a petri dish (diameter 9cm,
depth 1cm). Thimble filter papers (inner diameter 2.5cm, length 10cm), which were cut at a point 2cm
from the bottom, had a hole (inner diameter 2mm) at the bottom. After that the thimble bottoms were
turned over, and the termites and filter paper soaked in sample were covered with the thimble. All the
equipment was kept for 2h at room temperature. After 2h, the termites that emerged from inside of the
thimble filter paper were counted.

Identification of components

Identification of each component was done by gas chromatography-mass spectrometry (GC-MS) (HP,
6890-GC/5973- MSD). The GC column was a 30m • 0.25mm glass capillary coated with crosslinked
polyethylene glycol (film thickness 0.25/~m) (HP-INNOWax). The GC was programmed as follows:
started at 35~ held for lmin, then was increased from 35 ~ to 180~ at 6~ held lmin, increased to 250~ at
10~ and held for 20min. A splitless injection of 1/A was used. The carrier gas was helium (flow rate 1.0
ml/min). MS was EI mode (70eV). The peak was confirmed by comparison with a standard in the NIST
library data.

Preparation of the extract

Three hundred grams (300 g) from leaves powder was extracted with 300 mL of hexane (Fisher
Scientific, USA) in Soxhlet apparatus (Technico Scientific Company, Coimbatore, India) for 8 h. The
extract was filtered through a Buchner funnel with Whatman number 1 filter paper. The crude extract was
evaporated to dryness in a rotary vacuum evaporator. After complete evaporation of the solvent the
concentrated extract was collected and stored in a refrigerator at 4°C until required for assay. One gram
of the plant residue was dissolved in 100 mL of acetone (Fisher Scientific, USA) to make stock solution.
From the stock solution different concentrations; 25, 50, 75, 100 mg/L, were prepared.

Termite collection

Population of termites was collected from a termitarium from Arbaminch town, Arbaminch, Ethiopia and
identified in the Entomology Research Center. Termite mounds were dug up using shovel and soil
containing termites were kept in polyethylene plastic boxes. Termites were collected from the plastic
sheets using camel hair brush and placed in plastic containers as described by Addisu et al. (2013).
Termites were fed with dry wood inside the container and the top of the container was covered with
muslin cloth to allow free flow of air also to prevent the termites from escaping.

(lain sad ni nga study ang sunod ha pero collection gihapon :>)

Wooden blocks (20 X 4 X 2 cm) of kail wood were inserted into soil of termite infected area and made it
wet to maintain the moisture. After 10 days the wooden blocks were infested with termites and were
collected into plastic containers carefully with brush. Termites were kept in dark for 24 h at 28 ± 2 °C
temperature and 85 ± 5% relative humidity before experiments. Adult, healthy and active worker termites
were separated and used for bioassay.

Anti-termitic activity

The no-choice bioassay method of Kang, Matsushima, Sameshima, and Takamura (1990) was
employed to evaluate the anti-termitic activity of the plant extract. About 1 mL of plant extract of various
concentrations ranging from 25 to 100 mg/L were applied to Whatman No. 1 filter papers of 9 cm
diameter. Filter paper treated with acetone was used as a control. The solvent was removed from the
treated filter papers by air-drying at ambient temperature and batches of 20 worker termites were
randomly selected from the stock population and kept into respective Petri dishes (10 cm in diameter 1.5
cm in height). Treated and control termites were held under laboratory conditions in darkness at 27 ± 2°C
and 60–80% relative humidity. All treatments were replicated 3 times. The numbers of dead termites were
counted every 24 hours of exposure and the percentage mortality was calculated. A termite was
considered dead when it was lying flat on its back and showing no sign of body movement after being
touched with soft camel brush (mga bayooooooot, see dis huh?)

THE PROCESS
Ethanol extraction

The advantage to this approach is that the extraction is time efficient and of relatively low solvent-to-feed
ratio. However, the warm-ethanol technique is generally a small-batch approach that extracts
chlorophyll/waxes and decarboxylates the cannabinoids due to the heat involved. (Decarboxylation is the
conversion of THCA, for example, to THC through heating and agitation that yields carbon dioxide during
the process.) Therefore, heated ethanol extractions might require additional dewaxing and clarification
steps.
This type of technique is also limited in the number of products it can produce because all the acid-form
cannabinoids are decarboxylated during the extraction. While heating ethanol can increase the extraction
process’s efficiency, ethanol is a good solvent for extracting terpenes and cannabinoids.

In short, ethanol is a very good solvent as it applies to the extraction of cannabinoids and terpenes. In the
literature that describes the solubility of cannabinoids in ethanol, there is no definitive carry capacity, but
many sources suggest that cannabinoids are soluble in ethanol at a 1:1 ratio (meaning that 1g of THC is
soluble in 1mL of ethanol).

COMBINING ESSENTIAL OILS

Before beginning to create your own aromatherapy recipes of any sort, a good starting point is
to categorize your essential oils into groups that share similar traits. This can be by what they do (effects
you’re after), how they smell (scent type), or if you want to be really technical, you’ll sort them by their
chemical make-up of how fast each of them evaporates (notes).

In aromatherapy, mixtures of between two to five oils are most commonly used for blending, as that
seems to be the ‘sweet spot’ of achieving the most synergy between the oils. The success of your blend
will not only depend on simply mixing a few oils together, but also on which oils you choose to combine
and for what purpose.

PROPER WASTE DISPOSAL

2. All flies need to be terminated before final disposal. The preferred method is to place the insects in a -
20 °C freezer until no longer viable. The insects can be frozen in their primary containers and then
placed in the biohazardous waste receptacle or the biohazard bag containing the insects can be removed
from the biohazard waste receptacle, sealed, and placed in the freezer.
How Did I Get Flying Termites?

Also known as alates or reproductive stage termites, flying male and female alates emerge from existing
colonies to mate and form new nests elsewhere. A flying termite swarm near the home nest could
indicate a large colony in the yard or some other nearby location.

Swarms

Flying termites are visible when their colony swarms. Swarms are provoked by heavy rainfall and warm,
humid temperatures among other triggers. Swarms occur when established colonies produce winged
male and female termites in order to reproduce. After these mating flights, fertilized termites shed their
wings and go on to establish new colonies. Termites seen flying in a home are indicative of a mature
colony.

The bagged and cold-treated insect waste can then be disposed of in the same manner as other solid,
non-sharps biohazardous waste.

>In the Medical Center, place cold-treated bags in a secondary container inside your lab for pick up by
Environmental Services. (Assure that the Environmental Services representative that services your lab
knows where your waste will be stored for pickup.)

>If you have your waste collected for treatment and disposal by an outside contractor, place the bagged
insect waste into your contractor provided container for disposal.

Collection and Disposal of Insects Terminated in Alcohol

Insects collected in alcohol should not be placed in biohazardous waste for final disposal as flammable
substances should not be autoclaved. The container holding the alcohol and insect debris should be
leak-proof, wide mouthed, sealable, made of plastic rated to hold the alcohol used, and labeled as “insect
waste” with the alcohol solution present (70% ethanol). When ready for disposal, seal the container and
attach a completed pink chemical waste tag as you would for disposal of the alcohol.

Dear mga bayot,

Gitamad nako—charot. Mao ra bitaw na akong nakuha sa akong gibuhat na research : )

Nagmumura,

Eeghan alyas hakdog.