IWA Publishing 2012

Water Practice & Technology Vol 7 No 3

doi:10.2166/wpt.2012.050

Free-living microscopic organisms as indicators of changes in

drinking-water quality
Judit Plutzer* and Andrea Trkn
National Institute of Environmental Health, Gyli t 2-6, Budapest, H-1096, Hungary.
* Corresponding author. E-mail: plujud@yahoo.com

Abstract
Bacterial indicator organisms (e.g., coliforms, E. coli) and some chemical parameters (e.g., turbidity, ammonia) are
basic monitoring tools used to measure both changes in drinking-water quality and the presence of hard-todetect pathogenic bacteria, viruses and protozoa. Microscopically detectable free-living organisms, such as
some groups of bacteria, fungi, nematodes, rotifers and protozoa, are usable as additional indicators of fecal
or environmental contamination of drinking water as well as any changes in the drinking-water quality. Our
aim in this paper is to summarize the results of microscopic examination of 913 drinking-water samples from
different water sources in Hungary in 2004 and 2005 and to demonstrate how these results can be used to maintain safe and good-tasting drinking-water quality. A total of 277 drinking-water samples failed Hungarian
microscopic water quality standards as a result of helminths (58%), protozoa (41%), iron bacteria (16%), sulfur bacteria (13%), fungi (11%), algae (5%) and multiple biological contaminants (34%). Based on these results, pipe
washing or water storage tank cleaning was deemed necessary. In addition, a number of disinfection or ltration
failures were found. Two detailed case studies show the usefulness of monitoring microscopic parameters to
avoid disease outbreaks. To our knowledge this is the rst paper discussing drinking-water microscopy based
on Hungarian experience and practice, which could be useful and informative for other countries.
Key words: drinking-water quality, indicators, microscopy, monitoring

INTRODUCTION
The concept of using indicators as signs of fecal pollution is a well-established practice in the assessment of drinking-water quality (WHO 2008). An indicator parameter should provide evidence of the
presence or absence of a pathogenic organism that survives under similar physical, chemical, and nutrient conditions; however, it is important to distinguish between tests undertaken to signal the presence
of fecal pathogens or, alternatively, to measure the effectiveness of treatment processes (WHO 2008).
Any detectable coliforms in water supplies indicates inadequate treatment and can reveal regrowth and
possible biolm formation or contamination through ingress of foreign material (WHO 2008). The presence of Escherichia coli and intestinal enterococci provide evidence of recent fecal contamination.
Detection should lead to consideration of subsequent action, which could include further sampling
and investigation of potential contamination sources, such as inadequate treatment or breaches in distribution system integrity (WHO 2008). Important distinctions of intestinal enterococci include the
ability to survive longer in water environments than E. coli (or thermotolerant coliforms), as well as
increased resistance to desiccation and chlorination (WHO 2008). These indicator terms can also be
applied to non-microbial parameters; for example, turbidity can be used a ltration indicator. Increased
ammonia levels in water is an indicator of possible bacterial, sewage and animal waste pollution, and
the levels of total organic carbon (TOC) reect organic pollution stress. Finally, direct measurements of
residual disinfectant provide an indicator of disinfection efciency (WHO 2008).

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doi:10.2166/wpt.2012.050

Several studies have shown that the routine chemical parameters and bacterial indicators mentioned above are not 100% effective at identifying environmentally contaminated drinking water
(Plutzer et al. 2007; Kiss et al. 2008; Wilkes et al. 2009; Helmi et al. 2011). Routine detection procedures for total coliforms, E. coli, and intestinal enterococci mostly include membrane ltration,
incubation of the membranes on selective media at a specic temperature and colony counting
after 2448 h. This process results in time consuming detection of contamination because of the
lengthy incubation time. In addition, total coliforms and E. coli are signicantly more sensitive to disinfection than enteric viruses and protozoa, which makes these measures poor indicators of viral and
protozoan contaminants (WHO 2008).
The presence of Clostridium perfringens in drinking water can be an index of intermittent fecal contamination. In this case, potential sources of contamination should be investigated, and the detection
of C. perfringens in water immediately after treatment should lead to the investigation of ltration
plant performance. Because of the exceptional resistance of C. perfringens spores to disinfection processes and other unfavorable environmental conditions, the presence of this microorganism has been
proposed to be an index of enteric viruses and protozoa in treated drinking-water supplies. However,
the detection techniques of Clostridium are not as simple and inexpensive as those for other indicators, so this method cannot be used routinely (WHO 2008). Heterotrophic bacterial
measurements (HPC), ow cytometry and ATP measurements detect a wider range of microorganisms and are generally considered to be good indicators of distribution system integrity, which may
be compromised by stagnation or the development of biolms as well as a deterioration in cleanliness
(Hammes et al. 2008; WHO 2008; van der Wielen & van der Kooij 2010). The detection of HPC is
simple, but actual numbers are of less value than changes in numbers at particular locations, and there
is no evidence of an association of any of these organisms with gastrointestinal infection through
ingestion of drinking water in the general population (WHO 2008). The other methods (ow cytometry and ATP measurements) have similar values, although ow cytometry requires expensive
equipment (Hammes et al. 2008; van der Wielen & van der Kooij 2010).
Microscopically detectable free-living organisms, such as some groups of bacteria, fungi, nematodes,
rotifers and protozoa, are usable as additional indicators of fecal or environmental contamination of
drinking water as well as changes in drinking-water quality. Microscopy can provide inexpensive,
rapid results and reliable data on many drinking-water quality failures. The microscopy of drinking
water is an applied science that uses information from fresh water biology, or limnology. Microscopic
examinations of drinking water in Hungary started in 1895, when microscopy was rst performed by
Istvnffy (Felfldy 1984). The rst studies were mainly zoological or botanical examinations, and
only after the 1960s was the everyday practice of using indicator organisms detected by microscopy
introduced at water treatment plants in Hungary (Felfldy 1984). Drinking water analyses by microscopic biological test was rst published in 1977 to regulate these examinations (Hungarian
Standards Institution 1985). Routine microscopic water monitoring is included in the more recent Hungarian drinking-water legislation from 2001 (Hungarian Governmental Decree on Drinking Water
Quality and Monitoring 2001). Internationally, Hassall (London, England) was the rst to call attention
to the value of microscopic examinations in the interpretation of drinking-water analyses. About the
same time (1853) on the European continent, Cohn wrote his treatise Living Organism in Drinking
Water, in which he indicated the correlation between aquatic life and water purity (Whipple 1927). Previously, drinking-water microscopy was also practiced in the USA, England and a few other countries;
however, this method has fallen out of use (Whipple 1927) and it is used only in Central Europe (Slovakia and the Czech Republic) (cek et al. 1995; Sldeckov et al. 1996; Sldeckov 2000, 2002;
Ambrozova et al. 2003).
Our aim in this paper was not to prove that the commonly used bacterial and chemical drinkingwater examinations are necessary. Rather, our intention is to demonstrate the value and utility of
microscopic parameters, which are complementary to bacterial and chemical parameters. We

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doi:10.2166/wpt.2012.050

summarize the results of microscopic examination of 913 drinking-water samples collected from
different parts of Hungary between 2004 and 2005, and we indicate how these results can be used
to keep drinking water safe. Additionally, two case studies show the usefulness of these parameters
in the avoidance of water-borne infections.

MATERIALS AND METHODS

Sample collection for microscopic examination

One liter of drinking water was added to a clean glass bottle after rinsing the bottle with water from
the same source. Before sampling, the tap was allowed to run for 23 minutes. The samples were carried to the laboratory on ice and examined within 24 h.
The public water supply system covers all of Hungary and is based on several types of groundwater.
There are shallow, unconned aquifers; deeper, conned aquifers (used for drinking water down to
400 m in depth); and karstic aquifers. The so-called riverbank ltered aquifers follow multiple
rivers, including the Danube river, and these aquifers consist of gravel and sandy beds. The total
amount of water that originates from groundwater is 94.1% (conned ground water, 41.1%; unconned ground water, 2.6%; karstic water, 11.2%; riverbank ltrate, 39.4%), while 5.9% originates
from surface water. We selected sampling sites that covered the whole country and all of the water
types listed above. Overall, 913 drinking-water samples were collected in the time period spanning
20042005, and the samples represent 913 individual sampling points at the distribution systems.
Treatment of surface water includes occulation with alum, iron salts and/or synthetic organic
polymers; settling or sedimentation; sand or gravel ltration; and chlorination (with chlorine or chlorine dioxide) together with activated carbon ltration and, in some cases, ozonation.
The riverbank ltered and karst waters are directed to the drinking-water system after chlorination.
In a few cases the riverbank ltered water is directed to the water distribution system after treatment
that includes ozonation, sand and activated carbon ltration and removal of iron and manganese, in
addition to chlorination.
Hungarian aquifers (groundwater) are rich in iron, manganese, ammonium and arsenite/arsenate
ions. Therefore, treatments that remove these components are included in many places. The methods
that are used to remove iron and manganese include oxidation by aeration, followed by ltration or
employing oxidizing ltration media, which is coated with manganese oxide. Oxidation ltration
media has a catalytic effect on the chemical oxidationreduction reactions necessary for iron and
manganese removal, and the coating is maintained by regeneration with potassium permanganate.
Iron- and/or manganese-containing source waters that also contain arsenic are treated using conventional Fe/Mn removal processes. To remove As(V) in Hungary, ferric sulfate coagulation and
ltration is used. As(III) is converted through oxidation to As(V) using ferric chloride and potassium
permanganate. Breakpoint chlorination has been practiced as a physicalchemical process for removing ammonia. In several places, the recently introduced biological oxidation of ammonia to nitrite and
subsequently to nitrate is performed by the nitrifying bacteria Nitrosomonas and Nitrobacter and occurs
in uidized bed reactors.
Sample preparation for microscopy

A total volume of 990 mL of drinking water was ltered through a membrane (Schleicher & Schuell
PARATRADE; 0.45 m pore size, 47 mm, sterile). The seston (living organisms and particles of nonliving matter that oat in water and contribute to turbidity) was washed off of the membrane with a
sterile brush into 10 mL of the sample in a conical tube (calibrated to 0.11 mL) and centrifuged at

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doi:10.2166/wpt.2012.050

800 g for 15 minutes. The supernatant was aspirated off using clean Pasteur pipettes leaving 0.11 mL
nal volume, depending on the mass of the pellet. After homogenization, the resulting sample was
analyzed under the microscope (Leica DM 2500 or Leica DM IRB).
Determination of the quality and quantity of the pellet

Bioseston, identity of the organisms: All of the microscopically visible organisms (.3 m) were identied to determine the distinguishable taxonomic level at 4001,000
maximum magnication in the
absence of any preparation or dyeing. Sometimes, organisms were non-viable, immobile, in a dormant
state or the morphology of the cells was distorted or which made them unidentiable. In this case, the
microbes were grouped into a higher taxonomic category. We used several guidebooks that are available in different languages (English, German, Hungarian, Czech and Russian) in the international
literature to enable the identication of organisms. The selected guide books are available in the references (Bick 1972; Felfldy 1972; 1981; Cyrus & Sladecek 1973; Eikelboom & Van-Buijsen 1981;
Husler 1982; Wilken-Jensen & Gravesen 1984; Bancsi 1986; Foissner et al. 1991, 1992, 1994,
1995; Uherkovich 1995; Patterson 1996; Berger et al. 1997; Krammer & Lange-Bertalot 1997,
1999, 2004a,b; Nmeth 1997a,b; Csutorn 1998; Kiss Keve 1998; Komarek & Anagnostidis 1998,
2005; Schmidt & Fehr 1998, 1999, 2001; Grigorszky et al. 1999; Kiss Keve & cs 2004).
Viability of the organisms: Live algae uoresce red when excited with blue light. In the case of
motile organisms, live and dead cells can be distinguished according to the mobility of each microbe.
Organism viability is also of signicant importance in determining the disinfection efciency.
Number of organisms: Organisms were counted either in the total (0.1 mL) or in a part (0.02 mL or
3 rows/10 elds of vision across the coverslip) of the concentrated sample, depending on the expected
number of organisms. The number and volume of organisms counted were used to calculate the concentration of organisms in each sample, which were reported in individuals per liter (i/L).
Hungarian legalization: In Hungary, the statutory orders 201/200 (X.25.), 47/2005 (III.11.) and 65/
2009. (III. 31.) regulate the testing and examination of drinking water. Control microscopic monitoring is required once or twice per year at water treatment plants that process surface water, karst water,
groundwater (unconned aquifers) and riverbank ltered water. In other cases (conned aquifers),
microscopic monitoring is required every second year. The number of samples for microscopic analysis per water treatment plant is different and depends on the amount of water produced per day and
the number of settlements supplied. Microscopic examinations are mandatory after an incidence of
acute contamination, pipe repair, suspicious contamination, taste and odor problems, bad pipe conditions, biological problems, and high water temperature (.20 C), chemical oxygen demand
(COD(K2Cr2O7); .3.5 mg/L), total organic carbon (TOC; .2.0 mg/L) or NH4 (.0.5 mg/L).
Protozoa (agellates, ciliates, amoebae), helminths (nematodes, rotifers, Gastrotricha, tardigrades
and their eggs), sewage-pollution indicator bacteria (e.g., Zoogloea sp., Spirochaeta sp., Beggiatoa
sp.) and the conidia, spores or hyphae of fungi are required to be below the limit of detection. The
maximum allowable levels of the colorless, lamentous sulfur bacteria, known as Thiothrix spp. is
100 individuals (laments)/L and the allowable levels of iron bacteria is 20,000 individuals (laments)/L. The maximum allowable levels of algae and cyanobacteria is 10,000 individuals/L in
drinking water that is from surface water treatment plants and 1,000 individuals/L in other cases.
Although bacterial and chemical examinations are not the focus of this paper, and there are no statistically signicant relationships between the chemical, biological and bacterial results, we would like
to emphasize that routine chemical and microbial examinations were also performed in parallel with
the microscopic examinations based on the national legislation by the Decree of the Government No
201/2001 (X.25.). The Directive 98/83/EC of the Council on the quality of water intended for human
consumption has been transposed to this national legislation. The routine chemical and bacterial parameters that were tested and their standards are listed in Table 1.

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Table 1 | Summary of the routine drinking water examination parameters and the standards used in Hungary
Parameter

Unit

Detection limit

Standard

Heterotrophic plate count

22 C

CFU/mL

MSZ EN ISO 6222:2000 Water quality. Enumeration of

culturable micro-organisms. Colony count by inoculation
in a nutrient agar culture medium (ISO 6222:1999)

Heterotrophic plate count

37 C

CFU/mL

MSZ EN ISO 6222:2000 Water quality. Enumeration of

culturable micro-organisms. Colony count by inoculation
in a nutrient agar culture medium (ISO 6222:1999)

Coliform bacteria

CFU/100 mL

MSZ EN ISO 93081:2001 Water quality. Detection and

enumeration of Escherichia coli and coliform bacteria.
Part 1: Membrane ltration method (ISO 93081:2000)

Escherichia coli

CFU/100 mL

MSZ EN ISO 93081:2001 Water quality. Detection and

enumeration of Escherichia coli and coliform bacteria.
Part 1: Membrane ltration method (ISO 93081:2000)

Clostridia (not every

laboratory use
routinely)

CFU/100 mL

MSZ EN 264612:1994 Water quality. Detection and

enumeration of the spores of sulte-reducing anaerobes
(clostridia). Part 2: Method by membrane ltration

Pseudomonas aeruginosa
(not every laboratory
use routinely)

CFU/100 mL

MSZ EN 12780:2003 Water quality. Detection and

enumeration of Pseudomonas aeruginosa by membrane
ltration

Enterococcus (not every

laboratory use
routinely)

CFU/100 mL

MSZ EN ISO 78992:2000 V Water quality. Detection and

enumeration of intestinal enterococci. Part 2: Membrane
ltration method (ISO 78992:2000)

Chemical Oxygen
Demand K2Mn2O4

mg/L

0.05

MSZ 44820:1990 Drinking water analysis. Determination

of the chemical oxygen demand with potassium
permanganate

Ammonium

mg/L

0.01

MSZ ISO 71501:1992 Water quality. Determination of

ammonium. Part 1: Manual spectrophotometric method

Nitrite

mg/L

0.01

MSZ 148413:2009 Water quality. Part 12: Determination

of nitrate and nitrite. Content by spectrophotometric
method

Nitrate

mg/L

0.5

MSZ 148413:2009 Water quality. Part 12: Determination

of nitrate and nitrite. Content by spectrophotometric
method

Chloride

mg/L

MSZ 44815:1982 Drinking water analysis. Determination

of chloride ion

Sulfate

mg/L

12

MSZ 44813:1983 Drinking water analysis. Determination

of sulfate ion

Electrical conductivity

S/cm

MSZ 44832:1977 Drinking water analysis. Determination

of specic electrical conductivity

pH

MSZ 148422:2009 Water quality. Part 22: Detemination of

pH and pH in equilibrium state

Alkality

mmol/L

0.05

MSZ 44811:1986 Drinking water analysis. Determination

of basicity, hydrogen-carbonate ion, carbonate ion and
hydroxil ion content

Turbidity

NTU

0.05

MSZ ISO 7027:1992 Water quality. Determination of

turbidity

Total Hardness

CaO mg/L

0.5

MSZ 44821:1986 Drinking water analysis. Determination

of total hardness, carbonate hardness and non-carbonate
hardness

Iron

mg/L

0.01

MSZ 4484:1983 Drinking water analysis. Determination of

iron

Manganese

mg/L

0.01

MSZ 14842:1993 Water test. Determination of manganese

by spectrometric method

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RESULTS AND DISCUSSION

Results of the Hungarian drinking-water examination

During 20042005, 913 drinking-water samples were collected from different parts of the country and
examined. Based on the results, 277 (30%) of the drinking-water samples displayed inadequate microscopic water quality.
The samples were objectionable because of helminths (58%), protozoa (41%), iron bacteria (16%),
sulfur bacteria (13%), fungi (11%), algae (5%), or they contained multiple contaminants (34%). The
examined samples were deemed objectionable if any of the biological parameters listed above
exceeded the minimum values determined by our water quality standards.
Protozoan organisms were found in 144 of the examined samples. The most common organisms
were amoeboid cysts, Vahkamphia sp., Centropyxis sp., Euglypha sp., Trinema sp., Arcella sp., Peranema sp., and other agellates. Less common contaminants that were found included Mayorella sp.,
Litonotus sp., Cyclidium sp., Cinetochilum sp., Trachelophyllum sp., Aspidisca sp., Oxytricha sp. and
Colpoda sp.. Free-living amoebae, the motile, colorless agellates and ciliates are widespread in
environmental waters and consequently are present in both ground and surface waters that are
used for drinking-water production (Loret & Greub 2010). The prevalence of these microorganisms
and their concentration correlates with the level of organic matter present in the water, and their concentration is especially high in biolms and sediments, which constitute ecological niches where they
can feed on bacteria (Schiefner 1972; USEPA 2002; Loret & Greub 2010). The large amount of biolm mass present on the ltration media highlights the signicance of the colonization of granular
lters during drinking-water production, as well as the limited effectiveness of this technique in
removing these microorganisms (Sibille et al. 1998; Loret & Greub 2010). Due to the capacity of
some biolm residents to encyst, they are highly resistant to disinfection. Regrowth (excystation)
can occur in distribution systems, in which sediments, biolm and bacteria are also present in
large quantities (Loret & Greub 2010). Some free-living amoebae (e.g., Acanthamoeba sp., Hartmannella sp., Naegleria sp., Saccamoeba sp.) and ciliates (e.g., Tetrahymena sp., Cyclidium sp.) can act as
hosts for well-known and emerging pathogenic bacteria (e.g., Legionella sp., Mycobacterium sp., Salmonella sp., Yersinia sp., Shigella sp., Campylobacter sp.) and enable their protection and
dissemination in water systems (Sibille et al. 1998; Snelling et al. 2005; Bichai et al. 2008; Corsaro
et al. 2010; Loret & Greub 2010). In a recent review, a total of 102 well-known and 27 potential
pathogenic bacterial species were described as being competent to survive or grow within freeliving amoebae (Thomas et al. 2010). Some free-living Acanthamoeba spp. have been implicated in
diseases that result from contaminated drinking water. These Acanthamoeba spp. are pathogenic
and can cause corneal inammation, especially in people who wear soft or disposable contact
lenses, and have also been reported to cause chronic encephalitis and skin lesions in immunocompromised individuals (USEPA 2002). With regard to water treatment, control of free-living protozoa
cannot rely exclusively on disinfection. Removal processes including sedimentation, granular or membrane ltration constitute better options, with ultraltration being the best available technology
identied to date (Loret & Greub 2010). Control strategies of free-living protozoa should also address
the factors that favor their regrowth, or organic matter, biolms and sediments. Therefore, efcient
elimination of organic matter by clarication, good management of sludge from clariers, adapted frequency of lter backwash, and biolm and sediment control in distribution systems are all essential
measures to prevent the regrowth of free-living protozoa in drinking-water distribution systems
(Schiefner 1972; Loret & Greub 2010).
Helminths were detected in 162 samples. The most common organisms were Chaetonotus sp.,
Monostyla (Lecane) sp., Philodina sp., Bdelloidea sp., and Nematoda sp. Less common contaminants
that were found were Tardigrada sp., Aeolosoma sp. The presence of helminths in water distribution

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systems could be due to their survival and breeding during certain steps of the treatment process and
persistence during various stages of treatment, such as sand ltration (Adam et al. 1998; USEPA
2002). Rotifers and nematodes often colonize lters and are released into the lter efuents (Schiefner
1972; Adam et al. 1998). These organisms can also enter via reservoirs through vents and overows
(USEPA 2002). Certain species of nematodes can ingest pathogenic bacteria, such as Salmonella sp.
and Shigella sp., as well as Coxsackie virus and echovirus, and up to 16% of the ingested organisms
survive for 24 h at a temperature of 25 C inside their host (Chang et al. 1960; USEPA 2002). Locas
et al. (2007) conrmed the ndings described above, namely the ability of nematodes to protect bacteria. Furthermore, Caenorhabditis elegans (a model nematode) was reported to ingest, transport, and
excrete Cryptosporidium parvum oocysts (Huamanchay et al. 2004). The ingested oocysts remained
intact, viable, and infectious within the digestive tract of the nematode, with possible excystation
and freeing of sporozoites into the gastrointestinal system of the host. Nematodes that contained
oocysts and were exposed to desiccation for a day were able to cause an infection in mice, while
oocysts or nematodes having undergone the same treatment individually were not. Helminths have
eggs and cuticles that are resistant to chemical disinfectants (Bichai et al. 2008). High helminth numbers correlated with inappropriate lter quality, not enough backwash or water contamination, as
described above, and their presence was dependent on bacteria as a food source (USEPA 2002).
Microscopic examination detected lamentous fungi or their conidia in 30 samples. The most
common genera were Cladosporium sp., Alternaria sp., which have the potential to spoil food or beverages (Wilken-Jensen & Gravesen 1984). In the case of any uncertainties regarding the fungi species/
genera that are present in drinking water from the microscopic data, further mycological examination
(or examination of plate morphology) is advised. Based on the literature, other more harmful fungi
may also be present in the drinking-water distribution system, and some species have the potential
to cause allergic reactions or disease in humans, primarily in immunocompromised individuals
(e.g., Penicillium sp., Trichoderma sp., Aspergillus sp., Cryptococcus sp., Candida sp., Mucor sp., Stachybotrys sp., and Trichophyton sp.). Some fungi may also cause unwanted changes in the taste or
smell of the water (Phialophora sp., Acremonium sp., Penicillium sp.) (Frankov & Horeck 1995;
Doggett 2000; Gttlich et al. 2002; Hageskal et al. 2006; Sammon et al. 2010). The results from
our work, as well as several other studies, demonstrate that lamentous fungi and yeast are
common on water pipe surfaces or in water distribution systems (Frankov & Horeck 1995; Doggett
2000; Gttlich et al. 2002; Hageskal et al. 2006; Sammon et al. 2010). Low ow rates enhance biolm
development and therefore fungal growth. Pipeline cleaning and inspection of the water treatment
process (water stagnation, open storage tanks) should be considered when harmful microbiota are
detected in drinking water (USEPA 2002).
The number of iron bacteria in 45 samples was above the minimum recommended threshold of
20,000 laments/L. The most common organisms were Gallionella sp., Leptothrix species (including
L. ochracea and L. discophora), Crenothrix polyspora, and Clonothrix sp. Iron bacteria combine iron
or manganese dissolved in ground water with oxygen, and a side effect of this process is the production of a brown slime (Husler 1982). The slime does not constitute a health hazard, but it can
cause unpleasant odors and clog well screens and pipes. If the conditions are optimal, the bacteria
can grow at incredibly fast rates, and an entire well system may be rendered virtually useless in just
a few months (Whipple 1927).
Thiothrix (annulata) sulfur bacteria were detected at levels exceeding 100 laments/L in 35 of the
analyzed samples. These sulfur-oxidizing bacteria produce effects similar to those of iron bacteria by
way of sulde conversion into sulfate, which results in slime production. The sulfur bacteria are comprised of three groups that can be readily distinguished by microscopic examination: the green and
purple (or red) sulfur bacteria; the large, colorless, lamentous sulfur bacteria; and the large, colorless,
non lamentous sulfur bacteria (Husler 1982). In Hungarian drinking water, colorless, lamentous
sulfur bacteria were detected and restricted to places where H2S is present (Great Plain in Hungary).

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As a result of colorless, non lamentous sulfur bacteria exhibiting oxidative rather than photosynthetic metabolism, they can ourish only in regions where oxygen is available. Hence, they are
generally found in areas where one body of water with adequate oxygen content borders another
body of water that with signicant H2S levels. In this type of location, the colorless, lamentous
sulfur bacteria may form dense mats that are slightly yellowish-white in appearance (Husler 1982).
Iron and sulfur bacteria contribute to the corrosion of iron and steel well pipes and drinking-water
mains. These bacteria may cause corrosion by depleting dissolved oxygen, liberating corrosive metabolites, producing sulfuric acid from suldes or elemental sulfur and participating in the cathodic
process (USEPA 2002; llo s 2008). The best treatment for both iron and sulfur bacteria is growth prevention, so if their numbers exceed the minimum threshold value, pipe and/or system rinsing is
recommended (Schiefner 1972).
The number of algae in 15 of the analyzed samples exceeded the minimum threshold values. Microscopic examinations can provide information on the phytoplankton species present in water sources,
as well as their quality, and helps to explain the cause of turbidity and the presence of objectionable
odors (sh, grass or aromatic) and tastes in the water. Small sized Centrales Diatoms that are difcult
to remove by treatment processes persist in the treated water, and some lamentous green and bluegreen algae are also able to clog the lters (Schiefner 1972). If the algae appear viable upon excitation
with blue light then the disinfection was not effective. The presence of cyanobacteria, algae or other
surface water organisms in ground water is evidence of a direct inuence of surface water on the
ground water. Some species of cyanobacteria can cause skin symptoms, asthma and, if ingested, gastroenteritis, neuro-muscular disorders and liver damage; therefore, increased monitoring (toxin
measurement) is necessary at increased cyanobacterial levels (Chorus & Bartram 1999; USEPA
2002). The Water Safety Plan proposed by WHO mandates the determination of critical hazard
points during water production (Bartram et al. 2009). The biological parameters mentioned above
could be useful for analyzing the effectiveness of each step of the water treatment process and determining the best methods of purifying water that is enriched for algae as well as the efciency of
harmful cyanobacteria removal.
Based on the results above, most of the cases of contaminated drinking water required pipe washing
or water storage tank cleaning, although in some cases failures in disinfection or ltration were identied. After washing procedures were reinstated and the effectiveness of disinfection and ltration was
improved, water quality was continually examined until the relevant parameters met the Hungarian
standard requirements.
Case studies proving the usefulness of microscopy
Outbreak in the period of November 1324, 2004, in Nemesgulcs-Kisapti, Transdanubia, Hungary

Drinking-water pipelines were changed at the beginning of November 2004 in a wetland area close to
the supplied villages Nemesgulcs (998 inhabitants) and Kisapti (372 inhabitants). During the period
from November 13 to 24, 2004, a total of 203 inhabitants got sick after drinking tap water. The symptoms the inhabitants displayed were nausea, vomiting, diarrhea, and abdominal pain, and calicivirus
was detected in patient stool samples. Statistical analyses supported the high probability of infection
due to drinking contaminated water (Jzsa 2005). After the pipeline repair work, pipe rinsing, drinking-water disinfection, routine control examinations, which included bacterial and chemical tests, as
indicated in Table 1, and a microscopic test were performed. Although the caliciviruses could not be
identied by microscopy, the microscopic results identied a high number of helminths in the drinking water, establishing that the water was contaminated. Helminths were not present previously in the
drinking-water distribution system. Because the method of microscopy is very fast (results are available within 12 h, including the sampling step), this approach provided data indicating that the

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Table 2 | The impermissible parameters measured in the autumn of 2004 in the drinking water distribution system of the villages Nemesgulcs and Kisapti. After pipe repair work, the helminth number increased and reached very high
levels, which clearly showed that the pipe disinfection and rinsing after the completion of the repair work was
not effective. The water supply network was closed immediately after obtaining the biological results (12 h after
sampling) and helped to stop the disease outbreak, alleviating the prolonged length of time required for bacterial
results
Impermissible
bacteriological

Impermissible

parameters measured

Impermissible biological

chemical

Sampling period/Collected

(CFU, colony

parameters measured

parameters

Sampling sites

samples/Positive samples

forming unit)

(i/L, individual/liters)

measured

Nemesgulcs
drinking water
distribution
system

18.11.200422.11.2004.
23 samples collected (at 6
sampling points) 11 samples
positive (at 5 sampling points)

Coliform 1 CFU/
100 mL

Helminths 1300 i/L

Protozoa 250 i/L Iron
bacteria 200700,000 i/L

Kisapti drinking
water distribution
system

18.11.2004.22.11.2004
18 samples collected (at 5
sampling points) 11 samples
positive (at 4 sampling points)

Coliform 1 CFU/
100 mL Escherichia
coli 1 CFU/100 mL

Helminths 1800 i/L

Protozoa 7 i/L Iron
bacteria 38,800
900,000 i/L Algae
1,700 i/L

pipeline rinsing and disinfection was unsuccessful (data are summarized in Table 2), and the drinkingwater supply was closed immediately. This case illustrates that large drinking-water outbreaks can be
avoided through the establishment of fast action.

Outbreak during the period of June 516, 2006, in Miskolc, North Hungary

The catchment area of the karst springs (Italian well, New well) of city Miskolc is 76 km2. Between
May 23 and June 6, 2006, this area received 215.8 mm, or 16.8 million m3, of precipitation. The wastewater-containing pond Juhdglo overowed, and the pre-treated water (New well) of Miskolc was
contaminated by Juhdglo pond. The contaminated drinking water resulted in 3,673 of the 60,000
people living in the area becoming ill between June 5 and 16; of these 3,673, 161 patients were
admitted to the hospital. The patients symptoms were nausea, vomiting, diarrhea, abdominal pain,
and, in some cases, high temperature. Human calicivirus and Campylobacter jejuni and C. coli
were detected in the stool samples of 20 and 75 patients, respectively (Kiss et al. 2008). Statistical analyses supported the high probability of infection due to drinking the contaminated water supply (Kiss
et al. 2008).
Table 3 contains the impermissible parameters measured in the pre-treated water and drinkingwater distribution system of Miskolc in the spring of 2006. The data clearly show that unacceptable
biological and bacterial parameters were measured immediately before and during the disease outbreak period, in both the pre-treated water (New well) and in the drinking-water distribution
system. Although a large degree of turbidity was observed in the pre-treated water, this nding
alone did not prove that the well was contaminated because this high turbidity was repeatedly
observed without any microbial contamination problems every year following heavy spring rainfalls
in this area. The measurements of biological parameters indicated that the well was polluted with
ood surface water within 12 h after sampling (as a result of high levels of freshwater algae). The
most alarming bacterial indicators (E. coli and enterococcal bacteria) were available only after 24 h
of sampling. Only at later time points did the freshwater algae, E. coli and enterococcal bacteria
appear at some stages of the drinking-water distribution system, which were not obvious at earlier
sampling times. However, this outbreak could not have been avoided because of the delayed water
monitoring and response during the Hungarian public holidays.

Water Practice & Technology Vol 7 No 3

doi:10.2166/wpt.2012.050

Table 3 | The impermissible parameters measured in the spring of 2006 in samples of pre-treated water and of the drinking
water distribution system of Miskolc. Three samples were collected from the pre-treated water during this time (subsequent to these results the well was closed), and 230 samples were collected from different stages of the drinking
water distribution system to identify at which step(s) the water was contaminated. The biological analysis was the
rst method to identify the polluted surface water contamination of the well (i.e., the freshwater algae present in
the pre-treated well water)
Impermissible
chemical
parameters
Impermissible

measured

Sampling periods/Collected

Impermissible bacteriological
parameters measured

biological parameters
measured

(NTU,
Nephelometric

Sampling sites

samples/Positive samples

(CFU, colony forming unit)

(i/L, individual/liters)

turbidity unit)

Miskolc, raw
water, New
well

04.-09.06.2006. 3 samples
collected All samples positive

Coliform 200920 CFU/

100 mL E. coli 200
810 CFU/100 mL
Enterococcus 83 CFU/
100 mL Clostridium
32 CFU/50 mL

Algae 17,500 i/L

Pellet .0.1 mL/L

.10 NTU

Miskolc,
drinking water
distribution
system

23.05.-07.07.2006. 230 samples

collected for bacteriological
tests (at 60 sampling points)
18 samples for biological tests
(at 6 sampling points) 53
samples for chemical tests (at
19 sampling points) 8 samples
positive based on
bacteriological tests (at 6
sampling points) 6 samples
positive based on microscopy
(at 3 sampling points) 11
samples positive for turbidity
(at 8 sampling points)

Coliform 15200 CFU/

100 mL E. coli 15
200 CFU/100 mL
Enterococcus 283 CFU/
100 mL Clostridium 1
3 CFU/ 50 mL

Pellet 0.20.3 mL/L

Algae 17,500 i/L
Helminth 400
800 i/L

.2

CONCLUSIONS
Our ndings indicate that the results of chemical analysis and bacterial culturing do not substitute for
the information gained from microscopic analyses. Instead, these approaches complement each other.
Microscopic analyses of drinking water (in some cases, deposits, biolm and periphyton layers) help
to localize several areas of contamination in the drinking-water distribution system and indicate the
appropriate strategy to be employed to lengthen lter runs and prevent pipe difculty. The presence of
microscopic organisms in water provides valuable clues as to the origin of contamination.
The free-living microscopic organisms commonly encountered in drinking water do not cause disease in humans or in higher animals because they are not parasitic. There is no record of a specic
disease being traced to drinking water containing free-living microscopic organisms. However, the
bacterial, protozoan and viral reservoir function of the protozoa and helminths are considerable. Several authors recommend that free-living protozoa, helminths and fungi be removed from drinking
water. This method may be achieved by protecting the water source, implementing good practices
of water treatment, periodic and systematic swabbing and ushing of water pipelines and regular biological monitoring of water quality.
The so called water-borne diseases are caused by parasitic viruses, bacteria, protozoa and worms.
Some of these organisms can be studied under the microscope (e.g., Entamoeba histolytica, Giardia
intestinalis, Cryptosporidium spp., Acanthamoeba spp., Naegleria fowleri, Toxoplasma sp., Cyclospora
sp.), but they are seldom present in sufcient numbers in drinking water to nd them by routine
microscopy. Monitoring of specic bacterial, viral and protozoan pathogens is complex, expensive

Water Practice & Technology Vol 7 No 3

doi:10.2166/wpt.2012.050

and time consuming and may fail to detect their presence. It may take weeks to determine whether a
sample contains a particular pathogen. Thus, there is a great need to have fast detection methods to
prevent waterborne disease outbreaks, especially under unpredictable weather conditions (e.g., heavy,
storm rains, and oods). The microscopic organisms described in this study serve as valuable indicators of the environmental and waste water contamination of drinking water.
The microscopical drinking water examination method provides inexpensive, rapid and reliable
complementary data about drinking-water quality. Bacterial colony counting is time consuming detection of contamination because of the lengthy incubation time (over 24 h), while microscopic results
are available within 12 h and make possible fast action. As it is demonstrated at Nemesgulcs-Kisapti case study routine bacterial monitoring sometimes does not show heavy contamination problems.
Total coliforms and E. coli are signicantly more sensitive to disinfection than enteric viruses and protozoa, which makes these measures poor indicators of viral and protozoan contaminants, while freeliving microscopic helminths and protozoa have similar sensitivity to chlorine than enteric pathogens
mentioned above.
The only disadvantage of this microscopic method is the need for qualied people who are trained
in microscopy and microbial identication.

ACKNOWLEDGEMENTS
This work was supported by National Ofce for Research and Technology, Hungary, OM-00249/2008
and Healthy Water project EU FP62005-FOOD-4-B-36306. Authors would like to thank to Kroly
Jzsa, Zoltnn Kiss and to the Regional Waterworks of Nemesgulcs and Kisapti for help with
data collection.

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