Study design and location
The soil sampling was conducted at Kampung Orang Asli Sungai Lalang Baru, Ulu Semenyih, Selangor, Malaysia. The study location is about 22 Kms from the university’s laboratory (UniKL MESTECH, Malaysia). Some of the villagers practiced open defecation even now which resulted in soil contamination and soil transmitted helminthes (STH) propagations. The collections of soil sample were at the riverside, near the toilet area and at the pond areas.
Collection Of Soil Sample And Identification Of Soil Sample
The samples were collected by using a shovel to transfer the soil into a plastic bag prior transporting to the laboratory and used. Approximately 200–300 gram of soil were collected with a depth of 4–6 inch (optimized) in the area; common toilet area, near the riverside and near the pond area by using plastic bags and a small shovel. Upon reaching the laboratory the soil type was clearly labelled based on the location and stored in a refrigerator until used. Identification of type was carried out as previous described by Nisha et al, 2019 
Overall Ascaris culturing process
The Ascaris eggs were isolated and cultured by using three different process. The primary process was to isolate the Ascaris lumbricoides eggs using floatation technique, next step to culture the eggs with 0.1% sulphuric acid and finally to observe the embryonation stages of the Ascaris lumbricoides by using a light microscope.
Isolation of Ascaris lumbricoides
The first method for eggs isolation from soil sample is via flotation technique. Floatation technique was used to float up the eggs at the rim of the centrifuge tube. Next, McMaster slide was used to identify and to isolate the eggs.
For this, around 3 grams of the collected soil was mixed with distilled water (15 ml) in a centrifuge tube and centrifuged for 2500 rpm and for 5 minutes. The supernatant was discarded into the sink and filled up with 15 ml of the floatation fluid which was high density salt and/or sugar solution (specific gravity; 1.28).
Upon centrifugation, the centrifuge tubes were examined for any floating eggs. The eggs were observed at the edge of the centrifuge tubes and they were collected using a disposable pipette. Later, the eggs were transferred into the McMaster chamber to be observed under the light microscope for the count and morphology.
Culturing Ascaris lumbricoides
Around 1 ml of 0.1% sulphuric acid was used to culture the Ascaris lumbricoides. Upon confirmation of the egg’s morphology on the McMaster chamber, equal amount of Ascaris eggs were transferred into six different glass petri dishes. The ratio of Ascaris eggs in floatation fluid: volume of sulphuric acid was 5:1. The amount of the sulphuric acid volumes was optimized during the entire experiment.
Glass petri dishes were used instead of the normal plastic petri dishes because the sulphuric acid was highly corrosive therefore the plastic petri dishes was not recommended to due to reactivity. Next, the glass petri dishes were incubated at 37 °C for a few days and microscopic observation were carried out on daily basis up to 28 days, attributing to the life cycle of Ascaris lumbricoides embryonation.