Modification of the H₂S test to screen for the detection of sulphur and sulphate‐reducing bacteria of faecal origin in water

Abstract

The original H₂S test was developed in the 1980s to assess the microbial quality of
drinking‐water in low‐resource settings. This test was promoted as a promising
alternative to more sophisticated technologies, as the test can be performed in low‐
resource settings. However, the H₂S test lacks specificity for indicator organisms and has
never been evaluated by accepted method validation techniques. Despite these
criticisms, the H₂S test is a popular field test in many less‐developed parts of the world
and is promoted by many leading non‐governmental organisations because it is low‐cost,
easy to apply, and easy to read.
This PhD project investigated the performance of the H₂S test and its newly developed
modifications, by first establishing the detection threshold of the test and second, by
validating the H₂S test and its modifications against the internationally accepted
membrane filtration method. A final objective was to determine whether a media
formulation could be developed to detect Escherichia coli (E. coli). The diagnostic
sensitivity and specificity of the H₂S test was assessed by correlating its performance
against that of the modifications. All tests were incubated at 20, 37and 44°C to ascertain
operating parameters. All test variants were analysed against 20 pure‐cultured bacterial
species in order to identify those capable of triggering a positive result.
The research shows that it is possible to detect the faecal‐indicator E. coli by using an
adaptation of the original H₂S test. The five strains of E. coli were able to produce H2S in
the presence of organic sulphur compounds. The original H₂S test produced positive
reactions for Citrobacter freundii ATCC® 8090™, Proteus mirabilis ATCC® 43071™, and
Salmonella Typhimurium ATCC® 14028™ only. Modifications developed during this
research enhanced the sensitivity and specificity to E. coli when the source of sulphur was
changed from thiosulphate to L‐cysteine, L‐cystine, or 2‐mercaptopyridine. The inclusion
of bile salts, penicillin G, and L‐cystine increase the diagnostic sensitivity and specificity
for faecal coliforms. Furthermore, modified versions showed a comparable level of
diagnostic sensitivity and specificity (100%) with the standard membrane filtration
method using m‐FC media. The data presented in this thesis demonstrate that the H₂S
test variants developed during this research can be used as a screening test for the
assessment of microbial drinking‐water safety, in low‐resource settings.

Date of Award	Jul 2018
Original language	English
Awarding Institution	University of Brighton
Supervisor	Jonathan Caplin (Supervisor) & Ian Cooper (Supervisor)