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Hydrogen Sulfide (H2S) rapid field test for drinking water quality: feasibility
study in Tanzania

Technical Report · January 2016

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H2S rapid field test for drinking water quality:
feasibility study in Tanzania

Tanzania Integrated Water, Sanitation and Hygiene (iWASH) Program

H2S rapid field test for drinking water quality:
feasibility study in Tanzania
Funding for this publication was provided by the American people through the United States Agency for
International Development (USAID) as a component of the Tanzania Integrated Water, Sanitation and Hygiene
(iWASH) Program. The views and opinions of the authors expressed herein do not necessarily state or reflect those
of the United States Agency for International Development, the United States or Florida International University.

Copyright © Global Water for Sustainability Program – Florida International University

This publication may be reproduced in whole or in part and in any form for educational or non-profit purposes
without special permission from the copyright holder, provided acknowledgement of the source is made. No use of
the publication may be made for resale or for any commercial purposes whatsoever without the prior permission in
writing from the Florida International University - Global Water for Sustainability Program. Any inquiries can be
addressed to the same at the following address:

Global Water for Sustainability Program

Florida International University

Biscayne Bay Campus 3000 NE 151 St. ACI-267

North Miami, FL 33181 USA

Email: glows@fiu.edu

Website: www.globalwaters.net

For bibliographic purposes, this document should be cited as:

GLOWS-FIU. 2015. H2S rapid field test for drinking water quality: feasibility study in Tanzania Global Water for
Sustainability Program, Florida International University. 46 pp.

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Cover Photographs:
Contents

1. The hydrogen Sulphide test - a literature review

2. Lab trial results from MSABI
Low-cost field test for detection of bacteriological
contamination of water: hydrogen sulfide (H2S) paper strip
method in Tanzania
Amartya Saha,

Global Water For Sustainability Program

FIU

Summary: The primary goal in the provision of safe drinking water is that water be essentially
free of disease-causing microorganisms. The first step towards attaining this objective is to
periodically test drinking water at various stages from source to household consumption. Standard
tests recommended in Standard Water and Wastewater Treatment handbook are globally accepted
and performed; however their need of lab facilities, trained personnel and their cost limits their use
in rural and remote areas. In view of the strong need for testing in rural areas (for both local
communities and the protection of water sources for urban communities), as well as the tremendous
educational and decentralized ownership facilitated by a community performing their own tests,
several low cost, simple to perform tests have been developed over the past several decades, out of
which the hydrogen sulfide (H2S) paper strip test is been widely used in developing countries
worldwide. The UNICEF and WHO recommend the H2S test as the primary test for community
water monitoring for areas that do not have the benefit of an institutional testing program using
standard methods. The H2S test essentially detects the presence of hydrogen sulfide in a water
sample upon the formation of a black precipitate with iron; hydrogen sulfide is created by bacteria
associated with mammal intestinal linings and thus act as an indicator of faecal bacterial activity.
However hydrogen sulfide can arise from other sources as well, such as anerobic sediments and
geothermal vents, leading to false positives. This report reviews scientific publications examining the
efficacy of versions of the H2S method in comparison to established standard methods such as
membrane filter method as well as reports describing the implementation results across the
developing world, in order to collect current knowledge on this technique. It then includes a report
from MSABI describing lab and field trials of the H2S test in the Ifakara region. In the appendix is
included the list of reagents necessary to prepare these kits.
Drinking water quality monitoring: The need for affordable and easy
field tests for communities.

Testing for microbial contamination of drinking water

Waterborne diseases are one of the largest sources of infant and adult mortality in the developing
world. Hence, the most important goal for the provision of safe drinking water is that it be free of
disease-causing microorganisms. Determining the microbial quality of drinking water by measuring
the presence, absence or concentrations of indicator bacteria is universally practiced to: (1) meet
water quality standards and guidelines, (2) to determine source water quality, treatment system
efficacy and distribution system integrity, and (3) to inform Water Safety Plans, risk assessments and
management systems.
In addition, the measurement of the microbial quality of water for presence of fecal contamination
has other socially beneficial purposes. One such purpose is for community involvement and
ownership in the provision, management and monitoring of drinking water, including its sources
and treatment (Sobsey and Pfaender 2000). The ability of communities to test drinking water for
fecal contamination is a powerful and empowering tool for these purposes. Measuring the microbial
quality of water also serves an educational purpose. Making people aware about the links between
waterborne microbes/germs and disease within the context of education and outreach programs for
water, sanitation and hygiene at the individual, household, community and regional levels is a
continuing and long-term goal in health initiatives worldwide.
The availability of simple, practical, accessible and affordable tests for fecal contamination of
drinking water are extremely useful and potentially powerful tools, especially in the community
educational, monitoring and management context. In some situations the best tests to accomplish
these goals are those that are the simplest to use, understand, visualize and interpret. This is because
such tests can be widely disseminated both directly by the primary educators and then subsequently
via communications within households, families, schools and communities and by other means
(educational materials such as leaflets, signs and labels).

The necessity of field tests in rural areas and for communities

Methods to detect microbial contamination, as mentioned in Standard Water and Wastewater

Treatment are impractical for community level testing as they require laboratories, incubators,
specialized training/handling and time. The requirements for laboratory resources or costly field
analysis kits for standard bacteriological tests for fecal contamination of drinking water are major
barriers to their accessibility in many parts of the world. The need for sterilized bacteriological
materials (media, sample bottles, sterile diluent, culture tubes, bottle or plates, membrane filters,
pipettes or other volumetric dispensing devices, etc), controlled temperature incubators, the required
use of aseptic technique by trained individuals, and relatively high costs make it difficult, impractical
or impossible to perform these tests in many places. The resources and infrastructure are simply not
available to allow for routine bacteriological testing of drinking water using the standardized
methods for fecal indicator bacteria analysis.
The lack of availability of standard bacteriological tests for drinking water quality highlights the great
need for a rapid, simple, inexpensive test for the microbial quality of drinking water. This need is
especially great for small community and household water supplies that lack access to and can not
afford conventional bacteriological testing of drinking water. On-site testing using portable
equipment and use of simplified tests, such as the hydrogen sulfide (H2S) tests, may both contribute
to overcoming these constraints. For instance, Luyt et al (2012) report that the Colilert®18 system
that produces rapid results and is widely used in S.Africa produced false negatives (3.5% to 12.5%)
and that the Colilert medium has been reported to provide for cultivation of only 56.8% of relevant
bacterial strains. Identification of unknown sources of faecal contamination is not currently feasible.
Besides, it is also expensive, and therefore the authors recommend the H2S test.

The Hydrogen Sulfide (H2S) paper strip test

The H2S test was developed in India (Manja et al. 1982) and simply involves the addition of a water
sample (from tap, well or surface water body) to a clean bottle with a paper strip soaked with
specific reagents, and watching to see if the water sample turns dark brown to black over a 24 hour
period. This test is intended to detect bacteria associated with fecal contamination due to the activity
of these microorganisms in reducing organic sulfur to the sulfide oxidation state (as H2S gas) which
then reacts rapidly with iron to form a black, iron sulfide precipitate (Allen and Geldreich, 1975).
Some coliform bacteria (e.g.,Citrobacter spp.), some other enteric bacteria (e.g., Clostridium perfringens)
as well as many other types of bacteria produce H2S. Note that the H2S method does not
consistently measure the presence of either total coliform bacteria, specific groups of fecal bacteria
(e.g., fecal coliforms) or a specific fecal bacterium (E. coli) (Sobsey and Pfaender 2002); rather it
detects the presence of H2S in the water that always accompanies many faecal bacteria. Sobsey and
Pfaender (2002) review studies that describe a wide range of bacteria from both human and
domestic/agricultural animal faecal sources in water samples with positive reactions to the H2S test.

The advantage of the method is its simplicity, low cost and ability to be performed in the absence of
a typical microbiology laboratory or field laboratory test kit. Tubes or other containers holding the
test materials are prepared in a central laboratory to be used in the field by minimally trained
personnel. However, the presence of natural sources of H2S other than from bacterial activity, such
as from geothermal vents and anaerobic sediments in marshes can lead to a false positive result,
meaning that the test will detect H2S and indicate water is not safe to drink when there may be no
faecal bacteria. The number of false positives is of less public health concern when compared
against the speed, simplicity, and low cost of the test (Huang et al 2012).

Worldwide application:
The simplicity and good performance of the H2S method suggests that the method be tested in local
waters to examine for the possibility and extent of false positives in that region. With this objective,
over the last couple decades, various investigators have been testing this method and various
modifications of it in different tropical and temperate regions, including India, Indonesia, Peru,
Paraguay, Chile, West Indies, Pakistan, Nepal, Cambodia, Philippines, Fiji, Tanzania and South
Africa ( Ratto et. al., 1989; Kromoredjo and Fujioka, 1991, Kaspar et al., 1992; Castillo et. al., 1994;
Venkobachar et al., 1994; Ramteke et al 1994, Martins et. al., 1997; Rijal and Fujioka, 1998; Saeed et
al (1999). Genthe and Franck, 1999, Muller & O’Reilly 2002, Pant et al 2002, Tewari et al 2003,
Pathak & Gopal 2005, Izadi et al 2010, Everson (2011), Huang et al 2012), and compared it to
traditional bacterial indicators of fecal contamination of water. The results of these studies indicate
that the method gives results comparable to standard tests for traditional bacterial indicators of fecal
contamination. To illustrate this, a few recent study results are described below.
In Australia, Nair et al (2001) compared H2S results with results from standard procedures for
testing total coliforms, Escherichia coli and Salmonella spp, analysing 121 rainwater samples, 17 bore
water samples, 41 catchment water samples and 74 remote Aboriginal community water samples.
Rain water, bore water and catchment water samples gave true results of 78.5%, 82.3% and 80.5%
respectively while the treated and untreated community samples gave true results of 93.7 and 84.6%
respectively. It was concluded that in the developing countries where the acceptable level of total
coliform is <10MPN, the H2S method would be a good test to identify microbial contamination. In
other regions, the H2S method could be used as a screening test for drinking water supplies.

Similarly in India, Tambekar et al. (2008) carried out a study of 251 drinking water samples from 107
villages for potability of drinking water by standard accepted methods of water testing such as
Multiple Tube Fermentation Technique (MTFT) for determination of Most Probable number
(MPN), and Membrane filter techniques (MFT), Eijekman’s test for Thermotolerant coliform (TTC)
and Manja’s Rapid H S test for detection of fecal contaminations in drinking water. The efficiencies
of2 H S tests, MFT and TTC were 59 to 83%, 31% and 36% respectively when compared with
MTFT. Efficiency of H S test varies with the source and decreased with the depth of the source of
water. The study recommended that where there are laboratory facilities MFT and TTC and MTFT
tests should be performed, however, in fields and in villages, rapid and cheap H2S test should be
used for detection of fecal contamination in drinking water, for places where time, man and
laboratory facilities are very poor.
Pillai et al (1999) studied the minimum detectable level of faecal coliforms and the incubation period
required at various levels of contamination. The range of temperatures at which the method was
effective and the incubation period required at various temperatures were also determined. The H2S
method was found to be able to detect contamination down to a level of 1 CFU/100mL of coliform
bacteria. Although the H2S method could be used at a temperature range of 20 to 44oC,
temperatures between 28 to 37oC gave faster results. An incubation period of only 24 hours was
required at 37oC, which was found to be the most suitable incubation temperature. The incubation
period increased with a decrease or increase in temperature. In another study, Izadi et al (2010)
showed that the incubation period of H2S bottles is highly dependent on temperature and
concentration of faecal coliform bacteria, with shorter incubation times at higher temperatures; such
as 6 hours at 45 degrees C.

In Tanzania, Muller and O’Reilly (2002) carried out a study on 15 water samples from Lake
Tanganyika, 27 water taps in Kigoma and 4 water samples from the outlet of the water holding tanks
where treatment, disinfection by chlorine, occurs. Apart from finding the H2S test to be effective in
detecting bacterial contamination, their study points the importance of residual Chlorine in the
Kigoma municipal water supply that maintains low bacterial counts when water supply gets
contaminated from untreated groundwater and wastewater entry during power outtages ( when there
is no positive water pressure to keep external water out). 77% of samples with < 0.1 ppm residual
chlorine showed positive for H2S-producing bacteria, and only 7% of samples with 0.1 ppm or
greater residual chlorine were positive. These results agree with other studies in tropical countries
(Martins et al., 1997).

McNahan et al (2012) carried out a study to identify the types and numbers of microbial community
members present in natural water samples, including fecal indicators and pathogens as well as other
bacteria using biochemical and molecular methods and the Hach Pathoscreen-based MPN method
that is based on H2S detection. They found that H2S (+) samples also tested + for pathogens and
E.Coli, H2S (-) samples only one may have pathogens, and there were no E Coli (+) samples that
tested (-) for H2S. Their results further document the validity of the H2S test for detecting and
quantifying fecal contamination of water.

False positives

Although the intent of H2S tests is to detect bacteria associated with fecal contamination, there is
concern that the test also may detect bacteria not associated with fecal contamination and its
attendant pathogens. Therefore, an examination of the sources, sinks and transformation of sulfur
and the role of microbes in its cycling is important to understanding the applicability of this test.

There can be other sources of H2S in water apart from that produced by faecal bacteria. In
groundwater, particularly those contaminated with human or animal wastes or those containing
reduced sulfur from natural or anthropogenic sources, there is a high potential for anaerobiasis in
the aquifer and the resulting formation of sulfides by bacteria that are not of human or animal
origin. In addition, H2S producers may also be present in Iron-containing waters that contain other
Iron metabolizing and related corrosion bacteria. These bacteria may not have any relationship with
fecal contamination. Moreover, rapid reaction of the iron with sulfide already present in a water
sample could produce a darkening in an H2S test almost immediately upon addition of the sample.
(McMahan 2012).

Where sulfate concentrations in water are high, such as in geothermal environments, sulfate-
reducing bacteria could give positive results for fecal contamination in H2S tests. Sulfate reducers do
not metabolize complex organic compounds such as those included as substrates in H2S test
medium, instead requiring short chain organic acids and other products of fermentation. It is
possible that they would not grow and give positive results in H2S tests (Widdel, 1988)
because a test sample would need to become anaerobic to give a positive result and this is unlikely
during the 24-48 h test incubation time (Sobsey and Pfaender, 2002).

Many investigators have attempted to identify bacteria at the species level that produce positive
results in the H2S test (Sobsey and Pfaender, 2002). Castillo et al. (1994) found a variety of bacteria
of likely fecal origin in samples giving positive reactions in the H2S test, primarily Clostridium
perfringens and members of the Enterobacteriaceae (including Enterobacter, Clostridium,
Klebsiella, Escherichia, Salmonella, Morganella) and other organisms known to cause illness in
humans (Acinetobacter, Aeromonas). Ratto et al. (1989) found Citrobacter was a common organism
in positive H2S tests.
Sobsey and Pfaender (2002) suggest that while the organisms producing a positive H2S result many
not be all coliforms, they are organisms typically associated with the intestinal tracts of warm-
blooded animals, which are not necessarily of human origin
False negatives

Given the low solubility product of iron sulfide, the test can detect even small amounts of sulfide
formation or presence. However there are limits to detection; Huang et al (2012) mention that there
is the possibility of false negatives mainly resulting from the paper strip’s insensitivity to very low
bacteria count (i.e., < 100 CFUs). They suggest further studies to increase the sensitivity of the
strips, such as adding citrate to the H2S media.

Ease of use by communities

Luke et al (2004) assessed whether a simple paper-strip water-quality test (the hydrogen sulphide,
H2S) kit could be distributed and used for community-based monitoring following a disaster event
(cyclone) to better protect public health. The H2S test results correlated well with faecal and total
coliform results as found in previous studies. A small percentage of samples (about 10 per cent)
tested positive for faecal and total coliforms but did not test positive in the H2S test. It was
concluded that the H2S test would be well suited to wider use, especially in the absence of water-
quality monitoring capabilities for outer island groups as it is inexpensive and easy to use, thus
enabling communities and community health workers with minimal training to test their own water
supplies without outside assistance.

Huang et al (2012) reported that the diurnal temperature variation did not affect the overall reading
after three days. This suggests that sample bottles collected by community can remain stable in case
the observations are not made in the first 24 hours for any reason.

Conclusion

For any specific region, H2S tests deserve evaluation as accessible alternatives to conventional
bacteriological tests for fecal contamination of drinking water. Therefore, the potential merits and
beneficial uses of H2S tests deserve consideration, as does the determination of their reliability and
predictability in detecting fecal contamination of drinking water. Key issues to be addressed are
whether H2S tests are sufficiently reliable and adequately developed as tests of fecal contamination
of drinking water to be recommended for widespread and routine use, and if, so what caveats and
cautions should be applied and under what conditions. For these purposes the H2S tests and other
simple and affordable tests have great value and even greater potential use for drinking water supply
management and health education in the water and sanitation sectors. (Sobsey & Pfaender, 2002).
References

Castillo, G., R. Duarte, Z. Ruiz, M.T. Marucic, B. Honorato, R. Mercado, V. Coloma, V. Lorca, M.T.
Martins, and B.J. Dutka. 1994. Evaluation of disinfected and untreated drinking water supplies in
Chile by the H2S paper strip test. Water Resources. 8:1765–1770.

Everson J. Peters Water Quality of Rainwater Cisterns in the Grenadines

The West Indian Journal of Engineering Vol.33, Nos.1/2, January 2011, pp.56-64

Genthe, B. and M. Franck, 1999. A Tool for Assessing Microbial Quality in Small Community Water
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Forestry Technology, CSIR, Stellenbosch, South Africa.

Huang J, Zira J, Alkhelia D, Huang I and Veenstra J. (2012). Effectiveness of H2S strips for
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Izadi Morteza, Ahmad Sabzali2*, Bijan Bina2, Nematt A. Jonidi Jafari1,

Maryam Hatamzdeh2 and Hossein Farrokhzadeh. 2010.

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of drinking water in Indonesia, Env Toxicology and Water Quality
DOI: 10.1002/tox.2530060214
Luke M. Mosley Donald S. Sharp, Sarabjeet Singh. 2004. Effects of a Tropical Cyclone on the
Drinking water Quality of a Remote Pacific Island. Disasters, 2004, 28(4): 393–405

Luyt Catherine D. , Roman Tandlich , Wilhelmine J. Muller and Brendan S. Wilhelm. 2012.
Microbial Monitoring of Surface Water in South Africa: An Overview
Int. J. Environ. Res. Public Health 2012, 9, 2669-2693; doi:10.3390/ijerph9082669.

Manja, K.S., M.S. Maurya, and K.M. Rao. 1982. A simple field test for the detection of faecal
pollution in drinking water. Bulletin of the World Health Organization. 60:797–801.

Martins, M.T., G. Castillo, and B.J. Dutka. 1997. Evaluation of drinking water treatment plant
efficiency in microorganism removal by the coliphage, total coliform and H2S paper strip method.
Wat. Sci. Tech. 35:403–407.
McMahan Lanakila, Amy M. Grunden b, Anthony A. Devine c, Mark D. Sobsey. 2012. Evaluation
of a quantitative H2S MPN test for fecal microbes analysis of water using biochemical and molecular
identification. Water Research 46 (2012) 1693 - 1704

Muller P, Catherine OR. (2002). An examination of microbiological water quality in Kigoma,

Tanzania using a test for the presence of H2S producing bacteria, PhD. Thesis, University of
Arizona, 2002.
Nair J, R Gibbs, K Mathew and G E Ho. 2001. Suitability of the H2S method for testing untreated
and chlorinated water supplies Wat.Sci.Tech.44(6): 119-126

Pillai, J., Mathew, K., Gibbs, R. and Ho, G. E. (1999). H2S paper strip method-A bacteriological test
for faecal coliforms in drinking water at various temperatures, Water Science and Technology, 40(2): 85-
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Pant Hema, Sami Sarfaraz and Leela Iyengar. 2002. Evaluation of L-Cystine-amended H2S test for
the detection of faecal pollution in potable water: comparison with standard multiple tube
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Pathak S. P. and K. Gopal. 2005. Efficiency Of Modified H2S Test For Detection
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Annex 1

Commercial suppliers of H2S test lits

http://www.hach.com/pathoscreen-field-test-kit/product?id=7640249603
$49 for 100 tests, or 20 MPN tests

http://www.hach.com/pathoscreen-medium-presence-absence-powder-pillows-pk-
50/product?id=7640249608
$53 for 50 powder packs

http://www.hach.com/hydrogen-sulfide-test-kit-model-hs-c/product?id=7640219546
http://www.fondriest.com/hach-2537733.htm

http://www.hach.com/pathoscreen-medium-presence-absence-powder-pillows-pk-50/product-
downloads?id=7640249608

Suppliers in India

India field kits

http://www.indiawaterportal.org/node/1106

1.Development_Alternatives
DA manufactures H2S Vials/strip at its own Facility-TARA Environment Monitoring Facility,Delhi
under the name of "TARA-Aqua Check". DA has been in this business for 10 years and has
supplied in thousands to various customers in India and abroad. They have got the License from
National Research Developement Corporation(NRDC), New Delhi for its commercialization.
NRDC has made mandatory that suppliers/manufacturers of H2S strip kits/vials should obtain a
licensing certificate from NRDC, then take necessary training from DRDE-Gwalior for its
production/quality control, etc.
DA's address for contact:
DevelopmentAlternatives,
B-32TARACrescent QutabInstituonalArea,
New Delhi-110016,
Phone:011-26890380

Production site:
TARA-Environment Monitoring Facility,
TARA Nirman Kendra, Near NBCC Building,
M.G.Road, New Delhi-110030,
Phone: 011-26806172,26801521,26804482.
Cost of single vial: Rs.20+12.5% as VAT/CST+Rs.5 as Freight. Vials are sold in packs of 10.

2. WaterHealthLaboratories
Water Health Laboratories manufactures and markets these vials under contract with UNICEF.
The cost of one vial is Rs. 10/-. Orders must be made in batches of atleast 10 vials, for which the
charge is Rs 100/- + Shipping. The suggested payment is through DD with shipping cost payable on
delivery. The following contact information can be used:
Manufacturer's Contact:
Water Health Laboratories 4, Hydel Road, Industrial Area,
Roorkee - 247 667 INDIA
Tel: +91 (1332) 265320, 270053
Fax: +91 (1332) 274950
Cell: +91 94120 74490 (Dr. S.K.Sisodia)
Email: Click here

3. M/sLTEKSYSTEMS
For Chemical Parameter Kits, they provide sufficient reagents in Single and Multi-Parameter Field
Test Kits to carry out 100 nos. of test for each parameters. All the kits are based on visual colour
comparision method. The kits are portable, easy to carry any where, easy to operate, simple in use,
does not require any kind of energy or power. So simple in use that even a layman can also use it
comfortably. Does not require any technical support.
Manufacturer's Contact:
2-B, Rajkamal Complex, Panchsheel Square, Wardha Road, Dhantoli, Nagpur - 440 012
(Maharashtra),
Tel: (0712) 2442230 / 2706162
Fax: (0712) 2421746
Email: Click here
Contact Person: Mr. C. L. Lakhotia / Mr. Pramod Lakhotia.
Annex 2: Method of preparation of reagents for the Hydrogen Sulfide
(H2S) test

Saeed et al (1999) describe the preparation of H2S test kits that were made following the method
described by Manja et al. (1982). The media consisted of the following chemicals: 40g of
bacteriological peptone, 3g of di-potassium hydrogen phosphate, 2g of sodium thiosulphate, 1.5g of
ferric ammonium citrate, 2ml of liquid detergent and 100mL of de-ionised water. This provided
enough media for about 200 tests.

The strips were prepared by pipetting 2ml of the media on to the pads used to absorb broth used in
membrane filtration (47mm, Gelman) and were then dried at 50 °C in an oven. The dried pads were
cut into quarters to make four H2S strips each containing 0.5ml of the media. Each strip was then
placed in a clear plastic tube calibrated to 10ml volume, and sterilised by UV light in a biological
safety cabinet for at least 30 minutes. Alternatively, if pyrex bottles are used, a hot air oven or an
autoclave could be used for sterilisation. As long as the media impregnated paper strips are kept dry
and in the dark, they can be stored without refrigeration indefinitely. At the time of sampling, 10ml
water samples were collected directly into the H2S tubes, the lid tightly capped and the sample left in
the dark at ambient temperature (25–30 °C) for 36 hours.
After about 12 hours of incubation-time, and each 12 hours thereafter, an observation was made to
determine if a colour change had taken place. A change to black indicates a positive reaction and the
production of hydrogen sulphide, which typically indicates the presence of bacteria of faecal origin
(Manja et al., 1982; WHO, 2002a). The speed and intensity of the colour change is reflects the
density of sulphide-reducing bacteria present in the sample. The date and time of each
observation were recorded as follows: ‘–’ means no change; ‘+’ means slight change,
the paper strip or water has turned grey; ‘++’ means the paper strip is partially black;
‘+++’ means the strip and the water sample itself is noticeably black. The time-scale of
the colour change can also be interpreted in terms of risk to public health. A colour
change to black in less than 12 hours indicates a potentially high-risk situation, a
change on day two is associated with moderate risk and on day three, slight risk. At the
end of the field study, the samples were returned to a laboratory in Suva, the capital, for
analysis to determine the presence of Clostridium perfringens, an H2S producer and an
alternative indicator of faecal pollution. Although it was possible to test for its
presence a week after sampling due to it being a spore-forming anaerobic bacteria,
capable of surviving in the H2S media.

The H2S test

The procedure used to prepare the H2S culture media (M1 and M2), process the samples and
interpret the results were taken from (Manja, Maurya, & Rao, 1982); (Grant & Ziel, 1996); (Pillai,
Mathew, Gibbs, & Ho, 1999), (IDRC, 1998), and (Venkobachar, Kumar, Talreja, Kumar, & Iyengar,
1994). Furthermore, the originial medium established by (Manja, Maurya, & Rao, 1982)) used 1 mL
of Teepol. However, since Teepol is not widely available, (Grant & Ziel, 1996)) used lauryl sulfate
salts (or sodium lauryl sulfate) instead. Also, the H2S test reagent includes sodium thiosulfate, which
neutralizes chlorine present in a water sample. This means that the H2S test is a suitable
microbiological test for chlorinated water supplies
 H2S medium 40.0 g
Bacteriological peptone
Dipotassium hydrogen 3.00 g
phosphate
Ferric ammonium citrate 1.50 g
Sodium thiosulphate 2.00 g
Teepol 601/Sodium lauryl 0.20 g
sulfate
L-cystine (for M2 medium 0.25 g
only)
Water, distilled or boiled 100.0 mL
tap

The chemical composition for making of H2S strip is as follows:-

FOR MAKING OF 100ML OF SOLUTION FOR H2S STRIP:-

DISTILLED WATER 100 ML

FERRIC AMMONIUM CITRATE 1.5 GM
SODIUM THIOSULPHATE 2GM
SODIUM PYRATE 50MG
POTASSIUM THIOSULPHATE 3GM
L-CYSTINE 2.50GM
AGAR AGAR POWDER 30GM
SODIUM LAWING SULPHATE 200MG

Add 1ml of this solution to 80 sq. Cm of folded tissue paper. After this process the bottles are
capped properly and then they are auto claved for at least 1 hr, or till temperature of 210 degree C is
achieved. This step is not necessary if one uses boiled water and then can do with a low temperature
oven (Mosley 2005). Then a bottle is taken and a sample of water is tested in it for 24 hrs. A batch
of H2S strips is thus ready.
 FIU Subcontract Number: 800000246-05 (Amendment)

Water quality tests detecting hydrogen sulphide

producing bacteria for use in developing countries

Project Report

FIU Subcontract Number: 800000246-05 (Amendment)

MSABI Project Director: Dr. Niklaus Holbro

MSABI Project Administrator: Dr. Niklaus Holbro

Deliverables: Report stage 1

July 2013
Dr Jacqueline Thomas
Ms Fatuma Matwewe
 FIU Subcontract Number: 800000246-05 (Amendment)

EXECUTIVE SUMMARY

The results from the laboratory testing phase of the hydrogen sulphide (H2S) detecting
water quality tests are presented herein. Overall the reagents for the H2S test were able to be
sourced from suppliers in Dar es Salaam within reasonable timeframes and costs. The costs of
the reagents for a single test was only $US 0.28 with the recyclable glass bottles being the most
expensive component ($USD 5.33). The test was shown to detect to Salmonella enterica
(positive control) concentrations as low as 5 bacteria.100 mL-1 at ambient temperature. The test
was developed in a semi-quantitative form with a combination of 100 mL and 5 mL bottles to
give three risk ratings (safe, low and high risk). Against 20 environmental water samples the test
performed better (stronger and more rapid colour change) than against the lab positive control.
The first 24 h is the best time to read the test to show clear differences between improved (bores
with pumps, filtered water and tap water) and non-improved water sources (surface water and
open wells). At 48 h the tests develop further as the lower concentrations of bacteria grow up and
allowing for semi-quantification of the quality of the water source. It is recommended that the
tests be used in the field for water safety to the be read and demonstrated to the community
within the first 24 h. If more information about the water quality it needed then allowing the tests
to develop over 48 h will give the required level of detail. Based on the results from Stage 1 it is
recommended that the community use research outlined in Stage 2 of the proposal proceed as
outlined.

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 FIU Subcontract Number: 800000246-05 (Amendment)

TABLE OF CONTENTS

EXECUTIVE SUMMARY 1
TABLE OF CONTENTS 3
LIST OF TABLES AND FIGURES 4
1. INTRODUCTION 6
2. AIMS 6
3. METHODS 7
3.1 Positive control selection 7
3.2 Reagent sourcing and pricing 7
3.3 Media composition selection 7
3.3.1 Trials of media and temperature combinations 8
3.4 Efficacy of the test 9
3.5 Effectiveness with environmental water samples 10
4. RESULTS AND DISCUSSIONS 12
4.1 Reagent sourcing and pricing 12
4.2 Media composition selected 14
4.2.1 Media delivery method considerations 14
4.3 Efficacy of the test 15
4.4 Effectiveness against environmental water samples 17
5. CONCLUSIONS 22

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 FIU Subcontract Number: 800000246-05 (Amendment)

LIST OF TABLES AND FIGURES

TABLE 1. APPLICATIONS FOR THE H2S TEST FOR WATER SAMPLES FROM THE FIELD IN LESS DEVELOPED
COUNTRIES. ....................................................................................................................................................... 6

TABLE 1. SELECTION OF THE POSITIVE CONTROL MICRO-ORGANISM FOR H2S TESTS........................................ 7

TABLE 2. MEDIA COMPOSITIONS FOR H2S TESTS ................................................................................................... 8
TABLE 3. TRIAL OF MEDIA COMPOSITIONS .............................................................................................................. 9
TABLE 4. EFFICACY OF H2S TESTS WITH S. ENTERICA. ........................................................................................ 10
TABLE 5. ENVIRONMENTAL WATER SAMPLES TESTED. ....................................................................................... 11
TABLE 6. REAGENT SOURCING AND PRICING......................................................................................................... 13
TABLE 7. COST FOR REAGENTS AND BOTTLES PER TEST. WHERE ONE TEST INCLUDES A SINGLE 100 ML
SAMPLE AND A 5 ML SAMPLE. ....................................................................................................................... 13

TABLE 8. EFFICACY OF H2S TEST OVER A RANGE OF S. ENTERICA DENSITIES.................................................... 16

TABLE 9. RESULTS AND RISK CATEGORIES FOR SEMI-QUANTITATIVE H2S TESTS ............................................ 17

Figure 1. Reagents required to make the H2S media (central bottle with blue lid). 8
FIGURE 2. SOAKING FILTER PAPER WITH MEDIA .................................................................................................... 9
FIGURE 3. ENVIRONMENTAL WATER SOURCES SAMPLED. IMAGE A. MSABI ROPE PUMP. IMAGE B. TANIRA
PUMP. IMAGE C. SHALLOW OPEN WELL. IMAGE D. LUMEMO RIVER. ........................................................ 12

FIGURE 4. TRIAL OF MEDIA COMPOSITION. IMAGE A. 5 ML OF MEDIA WITH DETERGENT SHOWED DISTINCT
COLOUR CHANGE AFTER 24 H. IMAGE B. 2.5 ML OF MEDIA WITHOUT (LEFT) AND WITH DETERGENT

(RIGHT) AND DELIVERY OF MEDIUM BY SOAKED FILTER PAPER AFTER 7 DAYS AT AMBIENT
TEMPERATURE. ............................................................................................................................................... 14

FIGURE 5. TEST COLOUR CHANGE. IMAGE A. 100 ML BOTTLE COLOUR CHANGE RANGE. IMAGE B. 5 ML
BOTTLE COLOUR CHANGE RANGE. ................................................................................................................. 15

FIGURE 6. EFFICACY OF THE H2S TEST FOR 5 ML OF MEDIA AT AMBIENT TEMPERATURE AFTER 72 H.
NEGATIVE CONTROL IS THE BOTTLE ON THE FAR LEFT AND 1000 BACTERIA.100 ML-1 ON THE FAR
RIGHT. .............................................................................................................................................................. 17

FIGURE 7. MSABI ROPE PUMP WATER SAMPLES AFTER 24 H WITH H2S TEST BOTTLES AND MEMBRANE
FILTRATION PLATES IN FRONT....................................................................................................................... 20

FIGURE 8. OPEN WELL WATER SAMPLES AFTER 24 H WITH H2S TEST BOTTLES AND MEMBRANE FILTRATION
PLATES IN FRONT. ........................................................................................................................................... 20

FIGURE 9. FILTER POT WATER SAMPLES AFTER 48 H WITH H2S TEST BOTTLES AND MEMBRANE FILTRATION
PLATES PLUS AQUAGENX COMPARTMENTALIZED BAGS IN FRONT. ............................................................ 21

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 FIU Subcontract Number: 800000246-05 (Amendment)

FIGURE 10. TANIRA PUMP WATER SAMPLES AFTER 48 H WITH H2S TEST BOTTLES AND MEMBRANE
FILTRATION PLATES PLUS AQUAGENX COMPARTMENTALIZED BAGS IN FRONT. THE BLUE COLOUR

INDICATES A POSITIVE RESULTS FOR THE BAG TESTS. ................................................................................. 21

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 FIU Subcontract Number: 800000246-05 (Amendment)

1. INTRODUCTION

The H2S test has been employed in a number of different developing countries against a
range of water samples (Table 1). However, no literature could be identified were the test was
made or used in Tanzania. The test has been made in South Africa and used with success in the
field by community workers (Genthe and Jagals, 2003) which gives support to its
appropriateness for use in East Africa.
Table 1. Applications for the H2S test for water samples from the field in less developed
countries.

Country Area Samples Sample types Reference

Bangladesh Rural 382 Ground water, surface water, rain (Gupta et al., 2008)
water, stored water, ceramic pot
filtered water
Malaysia Rural 387 Ground water, piped water and (Desmarchelier et
stored water al., 1992)
India Rural 699 Piped water (Manja et al.,
1982)
Urban 90 Ground water, surface water, and (Pathak and Gopal,
piped water 2005)
Urban 1050 Ground water, surface water and (Tambekar et al.,
piped water 2007)
Urban 635 Ground water, surface water and (Hirulkar and
piped water Tambekar, 2006)
Indonesia Urban 46 Surface water and treated water (Kromoredjo and
Fujioka, 1991)
Vietnam Urban not Stored drinking water (McMahan and
provided Sobsey, 2010)
Fiji Rural 3 Surface water and rain water (Mosley and Sharp,
2005)
Peru Urban 20 Piped water (Ratto et al., 1989)

2. AIMS

The aims for this stage of the H2S research was to determine:
- if the test could be made in Tanzania;
- the cost of the test;
- the efficacy of the test against different bacteria densities; and
- the effectiveness of the test against a range of environmental water samples.

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 FIU Subcontract Number: 800000246-05 (Amendment)

3. METHODS

3.1 Positive control selection

There are a range of positive control organisms that have been used for the H2S test
reported in the literature (Table 2). Based on the growth requirements and likely availability of
cultures Salmonella spp. was selected as the best positive control options for this research.
Salmonella enterica subsp. enteria serovar Typhimurium UT30 (Gebreyes and Altier, 2002) was
sourced from the Department of Veterinary Medicine at the University of Sokoine, Morogoro.
The strain is a known to be multidrug resistant and was isolated from swine (Gebreyes and
Altier, 2002). S. enterica culture was isolated on the semi-selective MacConkey Agar and grown
in nutrient broth at 37 C for 24 h prior to inoculation.
Table 2. Selection of the positive control micro-organism for H2S tests.

Organism Considerations Reference

Salmonella spp. Most species produce H2S, grows on
MacConkey meida and non-selective
nutrient rich media.
Clostridium Spore forming organism and needs
perfringens anaerobic growth conditions
Klebsiella spp. Not commonly worked with
Citrobacter spp. Not commonly worked with
Escherichia spp. Only some spp, and strains produce
H2S
(Manja et al., 1982;
Castillo et al., 1994; Pillai
et al., 1999)
(Sobsey and Pfaender,
2002)
(Mosley and Sharp, 2005)
(Sobsey and Pfaender,
2002)
(Castillo et al., 1994)

3.2 Reagent sourcing and pricing

Suppliers in Dar es Salaam where contacted for pricing of the components required to
make the H2S tests. Prices were compared and the best value for money reagent selected. The
prices included shipping into Dar es Salaam and the associated Tanzanian Government
importation taxes. Price per unit test were based on the final media composition and quantity
selected.
3.3 Media composition selection
Based on the literature review two main variations in the media composition were
identified (Table 3). The main difference was the addition of liquid detergent. As the same brand
of liquid detergent (Teepol) could not be sourced efficiently in Tanzania a local brand (Jet,
Tarmal Limited Pty, Tanzania) was substituted (Figure 1). At this stage of the research the
specified enhancement additions such as L - Cystiene HCL and bile salts were not included. Also

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 FIU Subcontract Number: 800000246-05 (Amendment)

based on the literature the volume of H2S media required in each test varied from 2.5 mL
(Mosley and Sharp, 2005) to 5 mL (Manja et al., 1982) per 100 mL of water sample.
Table 3. Media compositions for H2S tests

Component Action Defence Centre for

Research and Disease Control,
Development USA
Agency, India
Bacteriological peptone Energy source 40 g 40 g
Dipotassium hydrogen Ion source 3g 3g
phosphate
Ferric ammonium Iron source 1.5 g 1.5 g
citrate
Sodium thiosulphate Sulphate source 2g 2g

Liquid detergent Surfactant 2 mL -

Water (sterile) Dilution 100 mL 100 mL

Reference (Manja et al., (Gupta et al.,
1982) 2008)

Figure 1. Reagents required to make the H2S media (central bottle with blue lid).

3.3.1 Trials of media and temperature combinations

Based on the different media compositions a matrices of tests were trialed to determine
the most effective combination (Table 4). The tests were trialed with autoclaved river water
(Lumemo River, Ifakara) spiked with 1 mL of 2 105 bacteria.mL-1 fresh S. enterica culture.
Negative controls were run as indicated. Tests were observed daily for a minimum of five days.

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 FIU Subcontract Number: 800000246-05 (Amendment)

Table 4. Trial of media compositions

Test Liquid Temperature Volume Delivery Negative

combination detergent incubated used method control run
in parallel
I Yes 37 C 2.5 mL soaked filter Yes
paper
II Yes 22 - 30 C 2.5 mL soaked filter
paper
III No 37 C 2.5 mL soaked filter Yes
paper
IV No 22 - 30 C 2.5 mL soaked filter
paper
V Yes 37 C 5 mL liquid Yes

Figure 2. Soaking filter paper with media

3.4 Efficacy of the test

To determine the efficacy of the H2S test and its detection ranges the tests were spiked
with known densities of S. enterica. Fresh S. enterica culture was grown as described and the
bacterial density calculated using a cell counting chamber (haemocytometer) and a light
mircoscope (Lieca). The experiment was conducted using a combination of bacterial densities
and conditions in two separate experiments. Media was added as liquid and made according to
Manja et al. (1982) including detergent. Bottles had 100 mL of sterile river water spiked with a
range of densities of S. enterica (Table 5) and incubated for five days. Additionally, for test
group II a 5 mL bottle was also included in the series and had proportionally 20 times less media
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 FIU Subcontract Number: 800000246-05 (Amendment)

(250 L) and S. enterica. The purpose of the 5 mL bottles was to see how the media performed
at smaller volumes to potentially develop a semi-quantitative test. Using a variety of bottles sizes
to achieve quantification has been reported for other H2S tests (Roser et al., 2005).
Table 5. Efficacy of H2S tests with S. enterica.

Test Media Temperature Estimated S. enterica bacteria density x 100

group volume mL-1
0 5 10 100 500 1000 10000
I 2.5 mL 22 - 30 C     
2.5 mL 37 C     
5 mL 22 - 30 C     
5 mL 37 C     
II 5 mL 22 - 30 C     
5 mL 37 C     

3.5 Effectiveness with environmental water samples

To evaluate the effectiveness of the H2S test with environmental water samples 20 water
samples were collected from around Ifakara and Lumemo (Figure 3). The H2S tests were
inoculated with 100 mL and 5 mL of water sample in duplicate and incubated at ambient room
temperature (22 - 30 C). The tests were read within one hour of inoculation to check that there
are no sulphide compounds already present in the water which may give a chemical induced
sulfide formation (Sobsey and Pfaender, 2002).
The 20 water samples were also tested for physical water quality parameters of pH (Hanna
Instruments) , salinity (ppm NaCl, HM) and turbidity (NTU, Nephelometer, Analite). In parallel
the 20 water samples were tested for Escherichia coli and total coliforms using membrane
filtration with m-ColiBlue24® growth media according to the US EPA 10029 method, except
they were incubated at 37 C. Additionally, the water samples were tested using a newly
commercialised field test for E. coli called the Aquagenx compartment bag test, which develops
at ambient temperature over 40 - 48 h (Aquagenx, 2013).

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 FIU Subcontract Number: 800000246-05 (Amendment)

Table 6. Environmental water samples tested.

Sample type Location Sample details Sample #

Tap water Ifakara IHI laboratory 4
IHI toilet block 20
Filter pot water MSABI house 2
IHI admin block 8
MSABI bores with MSABI House 1
rope pumps Mama Neema’s 9
Mama Benedict’s 10
Pottery 14
Tanira bores with Nazareti House 3
pumps Moyo 5
Mwakiwk 6
Kudinga 7
Shallow open wells Lumemo Opposite pottery 15
Tyre well 16
Next to marsh 17
Behind pottery 19
Surface water Lumemo river mid stream 1 11
Lumemo river mid stream 2 12
Lumemo river shallows 13
Marsh 18
IHI = Ifakara Health Institute

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 FIU Subcontract Number: 800000246-05 (Amendment)

Figure 3. Environmental water sources sampled. Image A. MSABI rope pump. Image B. Tanira
pump. Image C. Shallow open well. Image D. Lumemo River.

4. RESULTS AND DISCUSSIONS

4.1 Reagent sourcing and pricing

Reagents were sourced from various supplier in Dar es Salaam and priced. The average
length for importation from order was six weeks (Table 7). The cost of the test was calculated
using both a 100 mL and 5 mL sample volume. The reagent cost per water sample was
determined to be only $USD 0.28 (Table 8). The glass bottles are the most expensive purchase
($USD 5.33) but should last indefinitely if looked after and transported properly. The use of

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 FIU Subcontract Number: 800000246-05 (Amendment)

glass bottles is advantageous over disposable plastic because the media’s colour changes is
easier to see though glass, glass is can be autoclaved and recycling bottles reduces the amount of
waste produced by the test. Also, it is culturally appropriate in Tanzania as people are
accustomed to recycling glass bottles used for beer and soda.
Table 7. Reagent sourcing and pricing

Component Details Supplier Price TZS Price (USD)^

Bacteriological 500 g Lab Tech 95000 57.93
peptone
Sodium 500 g Lab Tech 45000 27.60
thiosulphate
Ferric 500 g Mainland 105950 65.00
ammonium Industries
citrate
Dipotassium 500 g Mainland 105950 65.00
hydrogen Industries
phosphate
Liquid Jet Local shops 7500 4.60
dishwashing 1L
detergent
100 mL glass 1 bottle Lab Tech 7335 4.50
bottle
5 mL glass 1 bottle Lab Tech 1353 0.83
bottle
TOTAL 368088 218.60
^ Exchange rate correct as at 2 July 13: 1 USD = 1630 TZS
Table 8. Cost for reagents and bottles per test. Where one test includes a single 100 mL sample
and a 5 mL sample.

Component Qty for 20 tests Cost / 20 tests Cost / tests USD

USD

Bacteriological 40 g 4.66 0.23

peptone
Sodium thiosulphate 2g 0.11 0.01

Ferric ammonium 1.5 g 0.20 0.01

citrate
Dipotassium hydrogen 3g 0.39 0.02
phosphate
Liquid dishwashing 2 mL 0.01 0.00
detergent

100 mL glass bottle 20 bottles 90.00 4.50

5 mL glass bottle 20 bottles 16.60 0.83

Sub total - reagents 5.64 0.28
Sub total - bottles 106.60 5.33

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 FIU Subcontract Number: 800000246-05 (Amendment)

TOTAL 112.24 5.61

4.2 Media composition selected
The media composition without detergent (Gupta et al., 2008) took > 72 h for the first
colour change to appear at 37 C while media with detergent (Manja et al., 1982) developed
within the first 24 h. At ambient temperature the media with out detergent did not develop at all
after 7 days of observation (Image B, Figure 4). Tests with 5 mL of media developed more
quickly (< 24 h) than 2.5 mL of media (> 48 h) at 37 C (Image A, Figure 4). The delivery
method of soaking filter paper in the media then adding it to the bottle was found to be time
consuming and unnecessary in a laboratory environment where the media could be autoclaved
after addition in liquid form. The best combination and volume of media was found to be 5 mL
with detergent delivered in liquid form into the bottles then autoclaved.

Figure 4. Trial of media composition. Image A. 5 mL of media with detergent showed distinct
colour change after 24 h. Image B. 2.5 mL of media without (left) and with detergent (right) and
delivery of medium by soaked filter paper after 10 days at ambient temperature.

4.2.1 Media delivery method considerations

Glass bottles with media could be prepared centrally then sent out to the field where the
tests are conducted then the glass bottles returned. The same process as for a bottle of soda to
reach a village and then the glass bottles returned and reused. The benefits of using the paper
delivery method are that the sterile dried paper impregnated with media can be simply
transported in a bag then added to sterile bottles in the field. The bottles are then reused and
sterilized in the field by boiling or baking in an oven. An alternative delivery means to
impregnated paper is having a prepared sterile bottle of media with a small graduated medicine
dropper. Small bottles of sterile media (10 mL) sufficient for 20 tests, could be prepared in a
central laboratory then distributed to the field. In the field 5 mL of the media can be dispensed
into bottles which can be reused and sterlised by boiling or baking in the field. Regardless of the

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 FIU Subcontract Number: 800000246-05 (Amendment)

delivery means the effectiveness of the test is still valid and the paper delivery method can be
easily adopted if chosen. However, without the paper the test is easier to read as there is a clear
colour change without any questions about what has happened to the white paper. Further, from
a behavioural perspective it is anticipated that the addition of the paper may warrant further
queries from the community about the reason for the black colour and was it the water or the
paper causing the change.
4.3 Efficacy of the test

Using the selected media composition (with detergent) the tests were trialed against a
range of S. enterica densities in liquid delivery at 2.5 mL and 5 mL. The first group of tests
showed that at ambient temperature that 2.5 mL of media took 96 h to have the first colour
change while 5 mL of media had the first colour change after 48 h. It was then confirmed that
5 mL of media was the best quantity to use for both time and the strength of the colour change.
The strength of the colour change was quantified for 100 mL and 5 mL bottles as detailed in
Figure 5. The 100 mL bottles had three distinct levels of colour change while the 5 mL bottles
had only two due to the smaller volumes.

Figure 5. Test colour change. Image A. 100 mL bottle colour change range. Image B. 5 mL
bottle colour change range.
Results from the second group of tests with 5 mL of media show that at incubated
temperatures (37 C) that the tests showed a distinct colour change after 48 h (Table 9). While, at
ambient temperature the test developed more slowly with distinct colour change observed after
72 h (Figure 6). The test was able to detect as few as 5 bacteria.100 mL-1 with colour change
observed being more gradual compared to the higher inoculums (1000 bacteria).

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 FIU Subcontract Number: 800000246-05 (Amendment)

Table 9. Efficacy of H2S test over a range of S. enterica densities.

S. enterica density 0 5 10 50 100 1000

Volume (mL) 100 5 100 5 100 5 100 5 100 5 100 5
Test Temp (C) Time (h)
Incubator 37 24            
group A 48            
72            
96            
Incubator 37 24            
group B 48            
72            
96            
Ambient 22-30 24            
group A 48            
72            
96            
Ambient 22-30 24            
group B 48            
72            
96            
 FIU Subcontract Number: 800000246-05 (Amendment)

Overall using the test at ambient temperature produced a distinct and observable colour
change after 72 h with S. enteria. The small volume tests (5 mL) were sensitive to `~
1 bacteria.5 mL-1 and will prove to be a useful cross check to the 100 mL volumes. As at one
twentieth the volume they will be able to differentiate between tests that have greater or less than
20 H2S producing bacteria.100 mL-1 when compared to the larger volume test (100 mL). This
will allow a risk category for the water source to be estimated as either safe, low or high (Table
10).
Table 10. Results and risk categories for semi-quantitative H2S tests

Levels 100 mL 5 mL H2S bacteria.100 mL-1 Risk

1 No change No change <1 SAFE
2 Black No change < 20 LOW
3 Black Black > 20 HIGH

Figure 6. Efficacy of the H2S test for 5 mL of media at ambient temperature after 72 h. Negative
control is the bottle on the far left and 1000 bacteria.100 mL-1 on the far right.

4.4 Effectiveness against environmental water samples

For the 20 environmental water samples the colour change for the H2S test was more
rapid and obvious (within 24 h) than that observed for the positive control S. enterica culture
(48 h) at ambient temperature. After 24 h there was complete colour change () for nearly all
the open well and surface water samples (Table 11 and Figure 8). While, the cleaner water
samples (taps, filter pots and bores with pumps) were all had no colour change except for one
Tanira pump (Figure 7). After 48 h the tests had developed and more of the improved water
sources had positive results. However, the cleanest samples, such as those from filter pots, did
not change (Figure 9). There was minimal change between the 48 h and 72 h readings.
 FIU Subcontract Number: 800000246-05 (Amendment)

Based on the risk categories for the H2S tests after reading at 24 h all the improved water
sources (except one Tanira pump) would be considered safe. While the open well and surface
water samples were all high risk (except one surface water sample). After 48 h more of the
improved water sources fell into the low and high risk categories as the lower concentrations of
H2S producing bacteria grew up.
The E. coli tests run in parallel directly support the H2S results. After reading the results
at 24 h the E. coli density from the membrane filtration results directly and significantly
positively correlated (r2 = 0.9, p < 0.0001) with the degree of colour change observed in the H2S
tests. Further after 48 h significant correlation was also observed (r2 = 0.79, p = 0.0003) but it
was weaker than for 24 h. The correlation between the H2S test across a range of water samples
gives confidence to its effectiveness for use as an indicator of feacal contamination. Further, it
must be remembered that H2S detects entirely different organisms to E. coli. Based on molecular
microbiology research is has been demonstrated that H2S producing organisms are a more
effective indicator of faecal contamination than E. coli (McMahan et al., 2012). For that reason
there is little merit in identifying false - positives or false - negatives between the two different
types of tests.
The Aquagenx compartment bag test took 48 h to change colour compared to 24 h for the
H2S test. The Aquagenx was complicated to use based on the how the media pellet is dissolved
(leaves a foam plug that needs to be removed), pouring the test into the bag and equally
distributing the sample between the compartments. Unfortunately, the tests were supplied
without sufficient clamps and therefore the bacteria moved between the compartments rendering
the test presence/absence only. The test is also expensive ranging form $6 - $10 per test
depending on how many tests are ordered (Aquagenx, 2013). Further, the test produces a lot of
plastic waste. On the positive side the test colour change was very easy to see with blue
indicating E. coli presence (Figure 10).
For the 20 environmental water samples the colour change for the H2S test was more
rapid and obvious (within 24 h) than that observed for the positive control S. enterica culture
(48 h) at ambient temperature. This is likely due to the variety of H2S producing bacteria present
in the water samples compared to the single lab culture of S. enterica used as the positive
control. It is also known that lab cultures grown on high nutrient growth medium take longer to
switch to a different carbon source (such as thiosulphate) than environmental bacterial which
may already be utilising less than ideal carbon sources to survive.

18
 FIU Subcontract Number: 800000246-05 (Amendment)

Table 11. Water quality and H2S test results from 20 environmental water samples.
Time (h) 24 48 72 RISK H2S
Test Physical H2 S E. coli H2 S E.coli H2S 24 h 48 h
pH TDS Turbidity 100 5 Membrane Bag 100 5 Bag 100 5
Group # ppm NTU A B A B Average Y/N A B A B Y/N A B A B
NaCl
Tap 4 7.8 212 0     0 N     N     SAFE LOW
water 20 7.2 223 4     3.5 N     N     SAFE LOW
Filter 2 8 199 0     0 N     N     SAFE SAFE
pot 8 7.5 222 0     0 N     N     SAFE SAFE
MSABI 1 7.5 196 43     9 Y     Y     SAFE HIGH
rope 9 7.2 808 0     0 N    N    SAFE SAFE
pumps
10 7.4 334 0     0 N    N    SAFE SAFE
14 7.6 101 7     0 N    N    SAFE SAFE
Tanira 3 7 215 0     0 N     N     SAFE HIGH
bores 5 7.6 254 0     1 N     Y     SAFE LOW
with
pumps
6 7.5 123 0     3.5 N     Y     LOW HIGH
7 7.3 161 0     2.5 N     Y     SAFE HIGH
Shallow 15 7.5 94 30     350 N     Y     HIGH HIGH
open 16 7.3 136 23     2850 N     Y     HIGH HIGH
wells 17 7.2 156 25     500 N     Y     HIGH HIGH
19 7.1 225 20     100 N     Y     HIGH HIGH
Surface 11 8.1 26.5 7     1050 N     Y     HIGH HIGH
water 12 7.9 25 7     1050 N     Y     HIGH HIGH
13 7.3 32.8 22     1050 N     Y     HIGH HIGH
18 7.2 171 19     100 N     Y     LOW HIGH
 FIU Subcontract Number: 800000246-05 (Amendment)

Figure 7. MSABI rope pump water samples after 24 h with H2S test bottles and membrane
filtration plates in front.

Figure 8. Open well water samples after 24 h with H2S test bottles and membrane filtration
plates in front.
 FIU Subcontract Number: 800000246-05 (Amendment)

Figure 9. Filter pot water samples after 48 h with H2S test bottles and membrane filtration plates
plus Aquagenx compartmentalized bags in front.

Figure 10. Tanira pump water samples after 48 h with H2S test bottles and membrane filtration
plates plus Aquagenx compartmentalized bags in front. The blue colour indicates a positive
results for the bag tests.

21
 FIU Subcontract Number: 800000246-05 (Amendment)

5. CONCLUSIONS

The reagents for the H2S test were able to be sourced from suppliers in Dar es Salaam
within reasonable timeframes and costs. The costs of the reagents for a single test was only
$US 0.28 with the recyclable glass bottles being the most expensive component ($USD 5.33).
The test was shown to detect to S. enterica concentrations as low as 5 bacteria.100 mL-1 at
ambient temperature. Against environmental water samples the test performed better (stronger
and more rapid colour change) than against the lab positive control. The first 24 h is the best
time to read the test to show clear differences between improved and non-improved water
sources. At 48 h the tests develop further as the lower concentrations bacteria multiply and the
full range of risk categories is apparent but the differences between the improved and
unimproved water sources less clear. It is recommended that the tests be used in the field for
water safety to be read and demonstrated to the community within the first 24 h. If more
information about the water quality it needed then allowing the tests to develop over 48 h will
give that level of detail. The strong correlation between the E. coli detection and the H2S test at
24 h and 48 h gives further confidence of its effectiveness at detecting faecal contamination.
Based on the results from Stage 1 it is recommended that the community research outlined in
Stage 2 proceed.

22
 FIU Subcontract Number: 800000246-05 (Amendment)

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23
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