Studies on Schistosoma haematobium... ( Cont.. 4)
MATERIALS AND METHODS
3.1 Study Area
This
study was conducted between March 2005 and June 2005 in Ile-Ife, Osun state by
examining urine samples collected from school-age children of two primary
schools for the presence of Schistosoma haematobium ova. Osun state
which is in South-western part of Nigeria is situated in the tropical
rainforest zone within latitude 7°28N and longitude 4°33'E.Climate is hot and humid with small seasonal and daily
variation. There is considerable variation in rainfall from year to year.
Portable water supply is inadequate and most parts still depend on natural
sources of water like streams, rainfall and wells for domestic purposes. (Udo
and Mamman, 1993).
3.2 Collection of urine samples
Prior to the collection of urine samples, the head mistresses
of the primary schools were
contacted for permission, co-operation and necessary briefing regarding the
purpose, relevance and personal involvement in the exercise. Questionnaires
were administered to the children and with the help of the school teachers,
pupil’s name, age, sex, occupation of parents, area of residence, sources of
water for bathing and domestic uses and history of terminal haematuria were
obtained. (Appendix III).
Specimen bottles were issued to
each of 99 pupils randomly selected for this study. And these specimen bottles
were labelled with pupil’s name, age and sex. All samples were collected
between 10.00h(10.00am) and 14.00h(2.00pm)- the period when maximum egg
excretion occurs or the peak of egg secretion (Cheesbrough, 1998) from pupils
who had just run around the school grounds after the break time to facilitate
the egg excretion. Visible evidence of haematuria was observed in some urine
samples.
3.3 Examination of urine
samples
After the collection of urine
samples, the specimen bottles were covered tightly and kept in dark containers
before getting to the laboratory to avoid hatching of the ova to release
miracidia on exposure to light. (Cheesbrough, 1998). However all samples were
taken to the laboratory immediately after collection. Sedimentation technique
was adopted for the examination of the urine samples (Richard et al.,
1984; Okanla, 1991).
Samples were allowed to settle for 30 minutes; by this time the schistosome eggs had settled at the bottom of the specimen bottles. Without disturbing the deposit, each of the supernatant urine samples was carefully poured away so that only 10ml were left. The content of each bottle was shaken to suspend the sediments. Two drops of undiluted formalin (formaldehyde) were added to each of the samples for preservation and to kill any emerging miracidium or miracidium that must have emerged. Afterwards, the samples were kept in a box and stored for considerable hours (of about 24 hours) in a cool place without any disturbance. Having done that, sediments of each urine samples clearly settled at the bottom.
This method was adopted when there was no centrifuging machine to use (Cheesbrough, 1998). However, when a centrifuging machine was available, 10ml of each urine samples that did not sediment clearly were resuspended and transferred into conical or centrifuging tubes then centrifuged at 1000rpm for 5minutes and sedimented. After discarding the supernatant, the sediment was resuspended in the remaining urine and poured into petri dishes (Anosike et al., 2001) and into counting chambers through the use of calibrated dropping pipettes for the examination of eggs of S. haematobium under the 10X objective of a binocular microscope. Eggs were counted and measured with the aid of eyepiece micrometer.
Samples were allowed to settle for 30 minutes; by this time the schistosome eggs had settled at the bottom of the specimen bottles. Without disturbing the deposit, each of the supernatant urine samples was carefully poured away so that only 10ml were left. The content of each bottle was shaken to suspend the sediments. Two drops of undiluted formalin (formaldehyde) were added to each of the samples for preservation and to kill any emerging miracidium or miracidium that must have emerged. Afterwards, the samples were kept in a box and stored for considerable hours (of about 24 hours) in a cool place without any disturbance. Having done that, sediments of each urine samples clearly settled at the bottom.
This method was adopted when there was no centrifuging machine to use (Cheesbrough, 1998). However, when a centrifuging machine was available, 10ml of each urine samples that did not sediment clearly were resuspended and transferred into conical or centrifuging tubes then centrifuged at 1000rpm for 5minutes and sedimented. After discarding the supernatant, the sediment was resuspended in the remaining urine and poured into petri dishes (Anosike et al., 2001) and into counting chambers through the use of calibrated dropping pipettes for the examination of eggs of S. haematobium under the 10X objective of a binocular microscope. Eggs were counted and measured with the aid of eyepiece micrometer.
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