The Hydrogen Sulfide Bacteria Test for Detection of Fecal Contamination of Water:
A Simple Test for Field Use
In 1982 Manja et al. reported the development of a simple method for detecting evidence of fecal
contamination in drinking water based on detecting hydrogen sulfide producing bacteria. The test
was intended to meet the "…need for a simple, reliable field test for use by village public health
workers" to detect fecal contamination in drinking water. They observed that the presence of
coliform bacteria in drinking water was consistently associated with organisms that produce
hydrogen sulfide (H2S). The test is based on the readily observable formation of an iron sulfide
precipitate on a paper strip (or in the water sample liquid) in a bottle or test tube, as a result of
the reaction of H2S with iron.
The H2S bacteria 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). Literally, the test measures the production (actually, the presence) of H2S by
its reaction with iron to form an insoluble, black precipitate of iron sulfide. Given the low
solubility product of iron sulfide, the test can detect even small amounts of sulfide formation or
presence.
There are concerns about the specificity of the test to detect fecal bacteria. This is because any
source of H2S in the sample can lead to a positive result. Sulfides also can be formed by abiotic
chemical reactions. Many different bacteria, from a variety of habitats, including many of enteric
origin, can release sulfide from proteins, amino acids and other reduced sulfur compounds by
reduction reactions. Therefore, there are many possible sources of a positive result in the H2S
test. Bacteria of possible fecal origin that produce H2S include various Enterobacteriaceae, such
as species of Salmonella, Citrobacter, Klebsiella, and Proteus, and sulphite reducing clostridia
such as C. perfringens. Some non-fecal bacteria that produce H2S are anaerobes belonging to the
genus Desulfovibrio.
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. Another major advantage is the simplicity and stability of the H2S bacteria
test media and the ability to pre-dispense the medium into sterile containers for storage and
transport and into which test waters can later be added directly to conduct the test.
Over the last two decades, various investigators have tested the H2S bacteria method and various
modifications of it in waters of different tropical and temperate regions. The test generally shows
reasonably good agreement with the detection of more conventional microbial indicators of fecal
contamination, and even with Salmonella bacteria. However, the ability of the H2S bacteria test
to predict the presence of other microbial pathogens or to predict human health effects (such as
diarrhea illness), has not been determined.
There are different versions of the H2S test, using slightly different culture medium, incubation
conditions and formats. The medium for the H2S bacteria test typically contains peptone, ferric
ammonium citrate, sodium thiosulfate, a detergent, L-cystine and phosphate buffer. The initial
test was for presence or absence of H2S producing bacteria in a specified volume of water, such
as 20 ml, using liquid broth culture. Later versions of the test have employed multiple sample
volumes and agar media to produced colonies as well.
PURPOSE
In this experiment we will analyze for H2S bacteria in selected samples of water and wastewater
using broth methods and different sample volumes to provide an end-point of sample positivity.
MATERIALS
Tubes of H2S broth medium for detection of H2S bacteria by a multiple volume method
Large tubes with 2 ml of medium for 20-ml samples
Small tubes with 0.2 ml of medium for 2-ml samples
The medium consists of (per 100 ml of reagent water): 40 g peptone, 1.5 g K2HPO4, 1.5 g ferric
ammonium citrate, 2.0 g sodium thiosulfate, 0.1 g sodium dodecyl sulfate and 0.025 g/L Lcystine, pH = 6.9
Milk dilution bottles of Standard Methods buffered water, 99 ml per bottle.
Pipets
Incubator, 35-37oC
Water and wastewater samples. To be specified by the instructor.
PROCEDURES
Work in pairs.
Sample Preparation and Dilution
Obtain a water and wastewater sample as designated by the instructor. The samples are raw
sewage, treated sewage effluent, upstream receiving water and downstream receiving water
(Morgan Creek). Each pair will analyze one wastewater (raw or effluent sewage) and one
receiving water (up or down stream Morgan Creek).
Shake samples 25 times immediately prior to inoculation into H2S broth or dilution.
Dilute up- and down-stream samples and sewage effluent 10-fold by adding 11 ml to 99 ml of
Standard Methods buffer in a milk dilution bottle. Shake 25 times
Dilute the raw sewage sample serially 10-fold through 10-7. Shake 25 times.
For up- and down-stream samples and sewage effluent, inoculate DUPLICATE large tubes with
20 ml of undiluted sample and inoculate small tubes with 2 ml of undiluted sample and 2 ml of
10-fold diluted sample.
For raw sewage, inoculate DUPLICATE small tubes of H2S broth with 2-ml volumes of the 10-3
through 10-7 sample dilutions.
For all samples, incubate one of the replicate culture tubes at 35-37oC and the other at ambient
(room) temperature.
After 24 and 48 hours, examine all tubes for H2S production, based on the appearance of a black
precipitate (black color in the medium or in the bottom of the tube). These are scored as H2Spositive sample volumes.
For samples incubated at room temperature, continue incubating negative tubes over the
weekend and examine for positivity on Monday (5th day of incubation).
Record all results for sample positivity and negativity in large and small culture tubes and bring
to class on the next Tuesday lecture period.
Results
Use the combined data for the whole class.
For each sample, record the numbers of large and small H2S broth culture tubes that were
positive and negative for H2S based on appearance of a black precipitate at each incubation
temperature (35-37oC and room temperature). Using the data for all replicate trials, compute the
MPN per 100 ml and the upper and lower 95% confidence limit values using the Thomas
equation. Remember to properly account for the sample volume per tube (20 ml for large tubes
and 2 ml for small tubes) and the sample dilution.
For each sample, determine if the MPN results for 35-37oC and room temperature incubation
agree. Do the MPNs at one incubation temperature fall within the 95% CIs of the other
temperature? Do the 95% CIs of the counts at the two incubation temperatures overlap? Does
one incubation temperature give consistently higher MPN concentrations than the other method?
What is the extent of H2S bacteria reduction by the sewage treatment plant? You can determine
this by calculating the relative amount of H2S bacteria in the effluent when the amount in the raw
sewage is considered 100%. This will give you the percent of initial indicator bacteria remaining
in the effluent.
What is the "background" level of H2S bacteria in Morgan Creek (the upstream level) and what
is the impact of the sewage treatment plant effluent on the Creek? This analysis requires you to
compare the levels of each indicator in the upstream water, the effluent and the downstream
water. That is, compare the absolute and relative concentrations of H2S bacteria in each sample
and explain the relationships among these H2S bacteria counts in the different samples?
REFERENCES
Allen, M.J., E.E. Geldreich. 1975. Bacteriological criteria for ground water quality. Ground
Water 13:5-52.
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.
Sobsey, M.D. and F.K. Pfaender (2002) Evaluation of the H2S Method for the Detection of Fecal
Contamination of Drinking-water. WHO/SDE/WSH/02.08, World Health Organization, Geneva.
At: http://www.who.int/water_sanitation_health/dwq/WSH02.08.pdf.