were stored at ambient temperature covered with material to keep out the
35 Compost was collected on the back of the health department's motor bike. Later a bicycle was
to facilitate easier movement around the island. Sometimes it was necessary to hitch a ride on
back of a utility if there was a larger amount of compost to collect. This illustrates one of the
'd1allenges which added to the complexity of conducting research in a remote location.
3 13
light. Solutions of 0.5% formalin and 0.1 NHz S04 were used to suspend the filtered compost samples and ova, and the tubes were stoppered with cotton wool to allow for
air
to enter the tubes. The formalin solution was diluted from10% formalin in saline solution that had been transported to Kiritimati
on a previous visit. Compost samples were prepared by filtering in the FPC tubes after shaking the sample in deionised water to which a few drops of Triton X detergent were added. The filtered material was then centrifuged at approximately 1 000 rpm for two minutes and the supernatant wasdiscarded. A small amount of 0.5% formalin or 0.1 NHz S04 was added to the sediment in the tube and inspected for
T.trichiura
ova under a Leica Galen 3 microscope.After inspecting the samples for ova, the tubes containing the ova were positioned vertically in a cardboard box with holes pierced in the top to accommodate the tubes. These were then incubated at ambient
temperature. Periodic checking was required to ensure that the samples had not dried out.
3.4.5
(b) Results of the viability analysis ofTrichuris trichiura
ova from the 1996 experimentsNo ova were observed in two of the compost/ soil samples so they were not prepared for incubation. Thirteen ova were detected in the soil/ compost Sample
1.
Ten of these were morphologically damaged in some way, and • three ova appeared to be undamaged. While the undamaged ova appearedintact morphologically, they contained no embryo. None of the ova in any of the compost samples contained embryo which meant that none of the ova were infective. This included the ova in Sample 13. However, since this was the compost used for the first pot trial bio-assay in April 1996 (refer section
4.8.1) it was considered important to incubate the samples in case there were
ova present so that the effectiveness of the locally made composting toilets in destroyingT.trichiura
ova could be assessed. Table3.9 records details of
samples that were incubated.All the samples listed in Table 3.11 were incubated for at least 24 days at ambient temperature . . Some were incubated for 30 days. However, no embryo developed in any of ova inspected in the 21 compost samples, one soil/ compost sample or the two control samples. This irlcluded th
�
control samples which made it impossible to assess the effectiveness of theincubation method. One example of each incubating tube was returned to I Australia in cabin baggage with other samples.
No. Date Solution No. of
No. 1 soil 14 months 1-5-96 H2SO4 I
No. 2A 1 1 months 26-4-96 fonnalin 1
No. 2B 1 1 months 26-4-96 H2SO4 1
No. 3A 1 1 months 23-4-96 H2SO4 1
No. 3B 7 months 23-4-96 H2SO4 1
No. 6A recent 1-5-96 H2SO4 1
No. 6B recent 2-5-96 H2SO4 1
No. 8A 1 1 months 26-4-96 H2SO4 1
No. 8B 1 1 months 26-4-96 fonnalin 1
No. 8C 4 months 25-4-96 fonnalin 1
No. 8D 4 months 25-4-96 H2SO4 I
No. IDA 1 month 25-4-96 H2SO4 1
lOB 1 month 26-4-96 H2SO4 1
10C 1 mqnth 26-4-96 H2SO4 1
lOD 1 month 27-4-96 Fonnalin 1
No. l2A 7 months 26-4-96 H2SO4 1
12B 1 1 months 26-4-96 H2SO4 1
12C 7 months 26-4-96 Fonnalin 1
120 1 1 months 26-4-96 Fonnalin 1
12E 2 weeks 1-5-96 H2SO4 1
No. 13A . 7 months 25-4-96 H2SO4 3
13B 7 months 25-4-96 H2SO4 3
Faecal fresh 2-5-96 H2SO4 1
Faecal fresh 4-5-96 H2SO4 1
TABLE 3.1 1 Samples prepared for incubation of Trichuris trichiura during April 1996 research visiL
The incubation was continued at the University of
Tasmania,
and new samples were prepared from compost and fresh faecal samples transported from Kiritimati. The samples were kept inFPC
strainer tubes that had been painted black and placed in an incubator which was kept at a constant temperature of30°C.
Three samples(IDA, 12E, 6A)
were simply mixed with Kiritimati soil and kept moist and incubated in an attempt to approximate the conditions normally experienced by ova in the soil. None of the ova £rom the compost samples or the control ova developed to the embryo stage indicating that the incubation procedure had failed.. Itis
possible that ova were affected by the ethyl-acetate used in the two phase extraction method which reportedlykill
or damageAscaris
ova (Viklund andDaIhammar
1996: 3).
Moody also argues that the addition of formalin or0.1%
sulphuric acid would renderT.tridlium
ova non-viable. as the shell isnot as resistant as
Asarris (pers.
comm. Moody17
December,1996).
It was concluded that further investigation of enumeration procedures for efficiently separating helminth ova from compost. and techniques for successfully incubating them, was necessaryif
the research on Kiritimati was to continue.Since none of the
T.tridzium
ova inspected in the1996
pathogen analysis contained any embryo they were not infective. However, it was important to knowif
theT.tridliura
ova still had the potential to embryonate and therefore be potentially infective.As
addressed in sections32
and3.2.2,
understanding the viability status of the ova would influence compost handling and usage procedures.It
would also indicate (in conjunction withspecific pathogen analysis) the possible presence or absence. of other categories of viable pathogenic organisms within the compost
No faecal coliform were detected in any of the
1996
Kiritimati compost samplestested
in Australia by microbiologist John Tranter at Richmond Pathology Service using the compost culturing method described in section3.4.6
(a). The method of collection, transport and storage of samples is detailed in section3.45
(a). Other planned pathogen testing and analyses of various materials transported from Kiritimati failed to eventuate as• described in section
3.4.6. After
obtaining funding for on-going research intothe Kiritimati project. a further research visit was organized from December
1997
to January1998.
The purpose of the visit was to continue theinvestigations begun in
1996.
This research included inspecting the condition and acceptance of the composting toilets, collecting samples to determine pathogens extant in the Kiritimati community, and to assess the extent towhich the composting toilets were inactivating pathogens entering the system.
3.45
(c) Viability analysis of1998
samples: isolation of ova using VISSar filter and incubation using Petrie dishesThe experience from the previous research indicated that a better method of filtering, isolating and enumerating the