Concentration method

The Midi Parasep faecal parasite concentrator, an enclosed system that employs the principle of the Ridley-Allen formol-ether sedimentation technique was used to concentrate samples for the comparison of ether and ethyl acetate [6]. A pea-sized amount (equivalent to approximately 1 gram) of faeces was mixed with 6 millilitres (mL) of 10% formalin in water in the mixing chamber. Two mL of ether or ethyl acetate was added (formalin/Triton-X 100 mixture was added to the mixture if ethyl acetate was used as it helps to emulsify the faecal matter). Parasep was assembled and sealed by screwing the filter thimble and sedimentation cone onto the mixing chamber. The mixture was vortexed for 15 seconds and the system inverted to allow the mixture to be filtered through the filter thimble (pore size of 425μm) and centrifuged at 1200g or 3000 rpm for 1–3 minutes according to the manufacturer’s instructions at the time. The mixing chamber and the filter thimble were unscrewed together and discarded. Like the conventional Ridley-Allen sedimentation method, there is an upper ethyl acetate layer, fatty plug, formalin supernatant and deposit. The fatty plug was loosened and the supernatant was safely discarded according to the Control of Substances Hazardous to Health regulations 2002 [7]. The concentration procedure apart from the centrifugation stage was undertaken in a Class 1 safety cabinet.

The Parasep faecal parasite concentrator with ethyl acetate as a solvent and Triton X 100 as a surfactant was used to compare the different centrifugal speeds and times. The recommended speed of 3000rpm and time of 1–3 minutes were altered according to the variations in speed and time quoted by participants.

The Parasep faecal parasite concentrator with ethyl acetate as a solvent and Triton X 100 as a surfactant according to the manufacturer’s instructions was used to compare 10% formalin diluted in water and 10% formalin diluted in saline as a preservative.

The conventional Ridley-Allen method with ethyl acetate and Triton X as a solvent was used to compare the sieves with different pore sizes.

Prior to the microscopic examination, all faecal deposits were re-suspended in 75μL of saline (three drops) and thoroughly mixed. In each case, 50μL of the diluted deposit was dispensed onto a microscope slide and a 22mm by 22mm coverslip applied.

The whole of the coverslip was examined and the number of ova, cysts and larvae recorded. All specimens were processed in duplicate and the mean calculated.