Vol.2, No.6, 582-588 (2010) Health
doi:10.4236/health.2010.26086
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Enumeration of microbial contaminants in sachet water:
a public health challenge
Narasimhan Banu*, Himabindu Menakuru
Department of Biotechnology, Vels University, Chennai, India; *Corresponding Author:
Received 18 November 2009; revised 8 March 2010; accepted 10 March 2010.
ABSTRACT
Accessibility and availability of fresh clean wa-
ter is a key to sustainable development and es-
sential element in health, food production and
poverty reduction. In the present study, we have
collected water sachet containing CM/L number
and they were analysed for physical and bacte-
riological nature. The organisms isolated in this
study were Proteus mirabilis, Klebsiella pneu-
moniae, Pseudomonas vesicularis and Pseu-
domonas aeruginosae. The harmful effects of
these isolates were evidenced by antibiotic re-
sistance, heavy metal tolerance and antibacte-
rial activity. They were resistant to the antibiot-
ics like amoxiclav, methicillin, chloramphenicol
and streptomycin. They showed tolerance to the
heavy metals at 5 mM conc. except for lead. For
antibacterial activity, they were tested against
human pathogens Klebsiella pnemoniae, Pro-
teus mirabilis, Micrococcus leuteus and Sal-
monella paratyphium. But at the same time
these organisms could be exploited for the in-
dustrial production of amylase, protease and
cellulase.
Keywords: Sachet Water; Bacterial Contaminants;
Pathogens; Industrially Useful Bacteria
1. INTRODUCTION
Bottled water is defined as water that is intended for
human consumption and this is sealed in bottles or other
container with no added ingredients except that it may
contain safe and suitable fluorides. Its price is within the
reach of tautology. Small scale entrepreneurs introduced
small nylon sachets which are electrically heated and
sealed at both ends to the market and is popularly called
pure water. It finds patronage from members of low
socio-economic class.
Besides, majority of the water sachets do not carry the
National Food and Drug Administration Control (NAFD
AC) approval number. This means that they are either
not registered or the procedures have not completed the
registration of their products with NAFDAC. It was
therefore considered implication of sachet water. Acces-
sibility and availability of fresh clean water is a key to
sustainable development and essential element in health,
food production and poverty reduction. 1.2 billion Peo-
ple around the world lack access to safe water and 2.5
billion are not provided with adequate sanitation (Third
World water forum on water, 2003). In metropolis as a
whole pipe born water is inadequate both in quality and
quantity.
Consequently water born diseases such as cholera and
typhoid often have their epidemic during the dry season.
Typhoid remains a great socio-economic problem in
developing countries. Proliferation of intestine is associ-
ated with high mortality with wound infection occurring
in 50-75% of surviours [1]. Controlling wound sepsis or
wound infection also affected mortality [2,3].
Since this health problem was largely traceable to
unhygienic water supply, an alternative to the seemingly
inadequate water supply was found in bottled water.
Drinking water including bottled water, may reasona-
bly to expected to contain at least small amounts of some
contaminants includes microbial pathogens, organic
pollutants like heavy metals. The presence of contami-
nants does not necessarily indicated that water poses a
health risk. Environmental Protection Agency (EPA) sets
standards for approximately 90 contaminants in drinking
water. EPA standards, along with each contaminant’s
likely source and health effects, are available at www.
epa.gov/safewater/mcl.html.
Microbiological contamination of water has long been
a concern to the public. There is a concern in increasing
due to outbreak of coliform bacteria, and protozoans like
Giardiasis, Cryptosporidiosis.
Coliforms are not a single type of bacteria but a group
of bacteria that includes Klebsiella, Proteus, E. coli, and
Salmonella. Coliform organisms are not necessarily

N. Banu et al. / HEALTH 2 (2010) 582-588
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
583
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pathogens and are rarely found in bottled water, they
serve as an indicator of insanitation or possible con-
tamination.
Microbial potability of bottled and packaged drinking
water hawked in Ilorin metropolis was done by selecting
81 samples containing 11 brands of drinking water
packaged and hawked in cellophane bags, did not met
drinking water standards. Pseudomonas was frequently
recovered as a contaminant of packaged water [4]. Bot-
tled mineral water consumption has significantly in-
creased in Brazil. Public health determines the parasi-
tological and microbiological status of some brands and
found occurrence of Cryptosporidial oocysts and
Guardia cyst in bottled mineral water [5]. An assessment
of the health and social economic implantations of sa-
chet water in Ibadam Nigeria [6] selected 78 samples
from 20 brands of sachet water from hawkers/vendors.
Bacteria obtained include: Klebsiella sp., Streptococcus
faecalis and Pseudomonas aeruginosae. By sterile filtra-
tion of water, broad diversity of viable bacteria was iso-
lated by using 0.2 μ filter for the removal of microor-
ganisms and is commonly referred as ‘sterile filtration’.
19 bacterial taxa were isolated by the acclimatization
method from 0.2 micron filtered fresh water samples.
Cryptosporidium parvum infection in Bergen and Nor-
way was found during the large water borne Giardiasis
outbreak [7].
The enforcement of the regulation guiding water qual-
ity before the National Agency for Food and Drug Ad-
ministration Control (NAFDAC) to employ with the
checking water qualities guideline values as recom-
mended by World Health Organization (W.H.O) be-
comes urgent.
The water sachets are the products of middle class en-
trepreneurs and some small scale business ventures. The
objective of the present study was to find out the quality
of sachet water. We have collected seven different
brands of water sachet containing CM/L number and the
samples were subjected to physical and bacteriological
analysis.
2. MATERIALS AND METHODS
2.1. Media Used
LB (Luria–Bertani) Agar, King’s B medium, Nutrient
Agar and EMB (Eosine Methylene Blue) Agar were used
to screen the sachet water sample for bacterial contami-
nation.
2.2. Collection of Samples
Seven sachet water samples supplied in and around Pal-
lavaram were collected. All of them contained CM/L
number along with ISI–14543, Ozonized and UV treated.
Some of them were Reverse Osmosis processed.
3. PHYSICAL PARAMETERS
pH: pH was checked for all the water samples immedi-
ately after opened.
4. BACTERIOLOGICAL ANALYSIS
ISOLATION AND IDENTIFICATION OF
BACTERIA
4.1. Isolation of Bacteria
Seven different sachet water samples were taken up for
the present study. The samples include Freeze, VSP,
VPZ, Aqua fresh, Jai, Hi-tech and Sakthi. The water
samples were serially diluted and spread on EMB me-
dium, Nutrient agar, King’s B medium and kept for 24
hours incubation at 37˚C. The isolated bacterial colonies
were purified to homogeneity by quadrant streaking,
store in LBA, NA and KBA slants periodically subcul-
tured.
4.2. Identification of Organism
The bacteria isolated were identified based on the bio-
chemical tests outlined in the Bergey’s Manual of deter-
minative bacteriology [8].
4.3. Antibiotic Resistance/Susceptibility
Screening
The sensitivity/resistance of the isolates to various anti-
biotics such as Chloramphenicol, Ceffriaxone, Amoxi-
clav, methicillin, Nalidixic acid and Streptomycin was
studied by inoculating a loopful of the overnight grown
cultures on Nutrient Agar plates amended with 30μg/ml
concentrations of the appropriate antibiotics and incu-
bated at 37˚C. After 24 hours of incubation, the plates
were observed for growth. Nutrient agar plates without
antibiotics served as control. The minimum concentra-
tion at which no growth was taken as the MIC (Mini-
mum Inhibitory Concentration).
4.4. Heavy Metal Tolerance Spectrum
The tolerance of the bacterial isolates to various heavy
metals such as zinc (zinc sulphate), lead (lead acetate),
copper (copper sulphate), chromium (potassium chro-
mate) was studied by inoculating loopful of overnight
grown cultures on Nutrient Agar plates amended with 1,
3 and 5mM concentrations of heavy metals and incu-
bated at 37˚C. After 24 hours of incubation, the plates
were observed for growth. Nutrient agar plates without
heavy metal served as control. The minimum concentra-
tion at which there was no growth was taken as the MIC

N. Banu et al. / HEALTH 2 (2010) 582-588
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value.
4.5. Screening for Extra Cellular Enzyme
Production
Four industrially important extra cellular enzymes were
selected and screened for primary extra cellular enzyme
production.
4.6. Protease
Nutrient agar along with 1% Gelatin (substrate) and 1%
casein (substrate) was taken in separate conical flasks
and autoclaved. They were poured into respective petr-
iplates (triplicates). After solidifying, isolates were stre-
aked on them and kept for incubation at 37˚C. After 24
hours of incubation, the plates were stained with 15%
HgCl2 (indicator) and observed for zone of inhibition.
4.7. Amylase
Nutrient agar along with 1% soluble starch (substrate)
was taken in a conical flask and then autoclaved. They
were poured into respective petriplates (triplicates). Af-
ter solidifying, isolates were streaked on them and kept
for incubation at 37˚C. After 24 hours of incubation, the
plates were stained with Gram’s Iodine (indicator) and
observed for zone of inhibition.
4.8. Cellulase
Nutrient agar along with 1% cellulose (substrate) was
taken in a conical flask and then autoclaved. They were
poured into respective petriplates (triplicates). After so-
lidifying, isolates were streaked on them and kept for
incubation at 37˚C. After 24 hours of incubation, the
plates were stained with 0.3% of Congo red (indicator);
plates were kept in orbital shaker (mild shaking) for 15
minutes. Congo red was discarded and then 1 N NaCl
was added to the plate and kept in shaker for 10 minutes
and observed for zone of inhibition.
4.9. Antibacterial Activity of Pure Bacterial
Isolates
Four bacterial isolates (Proteus mirabilis, Klebsiella
pneumoniae, Pseudomonas vesicularis and Pseudomo-
nas aeruginosae) from sachet water were selected for
antibacterial activity against the four human pathogenic
bacteria (Micrococcus luteus, Salmonella paratyphium,
Proteus mirabilis and Klebsiella pneumoniae) obtained
from CAS Botany, University of Madras. Isolated bacte-
ria were grown in King’s B Broth and kept for incuba-
tion at 37˚C in orbital shaking incubator for 48 hours.
After 48 hours incubation, samples were transferred to
sterile centrifuge tubes and centrifuged at 8000 rpm for
15 minutes at 4˚C. Then supernatant was collected and it
is filtered using 0.45 μ membrane filter. Filtered super-
natant was stored in refrigerator for further study.
Human pathogens as mentioned above were grown in
Nutrient Broth and incubated for 24 hours in orbital
shaking incubator at 37˚C. After 24 hours pathogens
were stored in refrigerator for further study.
Nutrient agar plates were prepared and pathogens
were swabbed on it and well were made on the plates
with the help of borer. 100 μl of filtered supernatant of
bacterial isolates was added into the wells and kept for
incubation for 48 hours. After 48 hours, plates were ob-
served for antibacterial activity.
4.10. Antibacterial Activity of Isolates with
Organic Solvent
The supernatant of the bacterial isolates as mentioned
above was taken. To that equal amount of Ethyl Acetate
(EA), an universally proved polar organic solvent that
could dissolve many compounds was added and kept in
orbital shaker for one hour or more preferably overnight.
Then transfer the organic layer and distribute it equally
in to different conical flasks and cover the flask with
cheese cloth to prevent contamination and after complete
drying, add EA. To the residue presenting the flask, the
supernatant was used for further study.
Human pathogens as mentioned above were grown in
Nutrient Broth and incubated for 24 hours in orbital
shaking incubator at 37˚C. After 24 hours, pathogens
were stored in refrigerator for further study.
Nutrient agar plates were prepared and pathogens
were swabbed on it and well were made on the plates
with the help of borer. 100 μl of filtered EA supernatant
was added into the wells and keep it for incubation for
48hours. After 48 hours, the plates were observed for
antibacterial activity.
5. RESULTS AND DISCUSSION
The objective of the present study was to find out the
quality of sachet water. For this, we randomly selected
seven different brands of sachet water and they were
collected from in and around pallavaram, Chennai. The
samples were subjected to physical and bacteriological
analysis. The water samples were assessed for coliform
and other bacteria using Nutrient agar, KB agar and
EMB agar. The results for this study support an earlier
observation that the sachet water being produced is of
questionable quality [9,10].
As useful as sachet water is to the society, the result of
the analysis raised doubts as to its quality. The pH of wa-
ter sachets has an upper range of 9.26 (Table 1), a value
higher than the upper limit of pH 8.5, as recommended
by W.H.O. Even though pH has no direct effect on
health, its indirect action on physiological processes

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Table 1. pH of the sachet water samples.
Name of the sachet pH
Freeze 9.20
VSP 9.26
VPZ 8.30
Aqua fresh 8.45
Jai 9.10
Hi-tech 8.38
Sakthi 8.20
cannot be over emphasized [6] got the upper pH unit of
9.7 among the 78 samples from 20 brands of sachet wa-
ter tested.
Bacteriological analyses results (Table 2) showed that
all the seven different brands of sachet water produced
growth after 24 hours of incubation. The organisms iso-
lated in this study are Proteus mirabilis, Klebsiella
pneumoniae, Pseudomonas vesicularis, and Pseudomo-
nas aeruginosae. [11] reported that pure water vending
machine may not be so pure, after all, because investiga-
tions found bacteria like E. coli in the machine. Isolation
of bacteria from pure water samples especially Strepto-
coccus facealis indicates possible contamination from
human excreta.
The procedure most often than not are those people
who may not know and are very little about the quality
of water sachets they produced. Some even imitate other
good products. For the price, pure water sachets are af-
fordable, to the middle class society and some of the
lower class also. Thus this type of packaged water is
now popularly and freely served at the common people
parties and social functions.
Most of the small scale producers of pure water may
not be able to afford the price or space for a bore hole in
their premises, hence they still depend on the already
condemned public water supply and water from doubtful
environmental sources, for the sources of the water they
use in packaging their products, some of them under
very poor environmental conditions. It should be noted
that the seven brands of sachet water selected for the
present study carried the CM/L number and ISI certified
but even they showed bacterial contamination. In the
present study, Pseudomonas was isolated from all the
samples and we are suggesting that the Pseudomonas
also be included as an added indicator for determining
their safety standard. Like us, Olayemi (1999) was fre-
quently isolated Pseudomonas from packaged waters.
Table 2. Biochemical characterization of the isolates from the sachet water.
Bacterial isolates
Biochemical test
Proteus mirabilis Klebsiella pneumoniae Pseudomonas vesicularis Pseudomonas aeruginosa
Colony morphology
Large, transparent, circu-
lar, raised, entire moist
colony
Small, non-pigmented
irregular lobate, raised
moist colonies
Small, pigmented, circu-
lar, flat, entire dry colo-
nies
Small, pigmented, circu-
lar, flat, entire dry colo-
nies
Gram’s staining Gram negative rods Gram negative rods Gram negative rods Gram negative rods
Motility Motile Non-motile Active motile Active motile
Catalase + + + +
Oxidase – – – +
Indole – – – +
Methyl red + – – –
VP test – + – –
Citrate test + + – +
Nitrate test + – – +
Starch hydrolysis – – – –
TSI
Alkaline slant, acid butt,
H2S positive, No gas
production
Acid butt, alkaline slant,
No gas and H2S produc-
tion
Alkaline butt, acid slant,
No gas and H2S produc-
tion
Alkaline butt, acid slant,
No gas and H2S produc-
tion

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The isolates of the present study (Proteus mirabilis,
Klebsiella pneumoniae, Pseudomonas vesicularis and
Pseudomonas aeruginosae) were tested for their antibi-
otic resistance against ceffriaxone, nalidixic acid,
amoxiclav, methicillin, chloramphenicol and streptomy-
cin (Table 3). All of the isolates are sensitive to ceffri-
axone and nalidixic acid and are resistant to amoxiclav,
methicillin, chloramphenicol and streptomycin. That is,
their presence in the water sachet pose a danger for those
who are taking. The consuming public also must be in-
formed of the consequences of consuming packaged
water. Even though the water sachets are UV-treated and
ozonized, it could be also recommended that produced
packaged water should endeavor to disinfect their prod-
ucts with solar radiation, which is simple to construct
and easy to maintain.
Proteus mirabilis, Klebsiella pneumoniae, Pseudo-
monas vesicularis and Pseudomonas aeruginosae were
tested for their heavy metal tolerance. For this study, we
selected only four heavy metals namely, copper (copper
sulphate), chromium (potassium dichromate), zinc (zinc
sulphate) and lead (lead acetate) at a concentration of 1
mM, 3 mM and 5 mM. All of the four isolates showed
growth at 1 mM and 3 mM concentration. But at 5 mM
concentration, the heavy metal except lead seems to be
toxic to the isolates and no growth was observed. This
result indicates that these organisms are resistant to
heavy metals at 1 mM and 3 mM concentration. For lead,
they are showing tolerance even at 5 mM concentration
(Table 4).
We screened three of the industrially important extra
cellular enzymes (protease, amylase and cellulase) pro-
duction by our test organisms. For protease enzyme, two
different substrates were used as nitrogen source i.e.,
Gelatin and casein. All the test organisms showed prote-
ase production by plate assay method. For amylase pro-
duction, starch served as a sole carbon source and the
Proteus mirabilis and Klebsiella pneumoniae is the pro-
ducer of the sole carbon source but this was not pro-
duced by any of the organisms tested. The isolates of
sachet water shows some harmful (antibiotic resistance,
heavy metal resistance) and useful properties. Useful
property includes industrial enzyme production. So we
can exploit these organisms for the mass production of
industrial enzymes like protease and amylase (Table 5).
Among four bacteria Klebsiella pneumoniae and Pro-
teus mirabilis were considered as human pathogens. To
know its antibacterial property, we selected four human
pathogens like Klebsiella pneumoniae, Proteus mirabilis,
Micrococcus luteus and Salmonella paratyphium. Pro-
teus mirabilis and Klebsiella pneumoniae showed anti-
bacterial activity against Micrococcus luteus and Sal-
monella paratyphium. Pseudomonas vesicularis showed
no antibacterial activity against Proteus mirabilis, Mi-
crococcus luteus and Salmonella paratyphium but it
formed zone of clearance against Klebsiella pneumoniae.
Pseudomonas aeruginosa showed antibacterial activity
against Proteus mirabilis and Salmonella paratyphium
but no activity for Klebsiella pneumoniae and Micro-
coccus luteus (Table 6). The same result was observed
for ethyl acetate fractions of bacterial isolates except
Proteus mirabilis showed antibacterial activity against
Klebsiella pneumoniae (Table 7).
Since Proteus mirabilis and Klebsiella pneumoniae
itself was a human pathogen, it does not showed anti-
bacterial activity against the same human pathogen
brought from outside. But we could not exploit this to
control Micrococcus luteus and Salmonella paratyphium.
Pseudomonas vesicularis showed activity against Kleb-
siella pneumoniae and Pseudomonas aeruginosa showed
activity against Proteus mirabilis and Salmonella para-
typhium. We could also exploit these two isolates against
Table 3. Antibiotic resistance/sensitivity spectrum of isolates from sachet water.
Bacterial isolates
Antibiotic Concentration μg/ml
Proteus mirabilis Klebsiella pneumoniaePseudomonas vesicu-
laris
Pseudomonas aerugi-
nosa
Ceffriaxone 30 + + + +
Chloramphenicol 30 – – – –
Amoxiclav 30 – – – –
Methicillin 30 – – – –
Nalidixic acid 30 + + + +
Streptomycin 30 – – – –
+  sensitive
–  resistant

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Table 4. Heavy metal tolerance spectrum of isolated strains from sachet water.
Bacterial isolates
Heavy metals Concentration μg/ml
Proteus mirabilis Klebsiella pneumoniaePseudomonas vesicu-
laris
Pseudomonas aerugi-
nosa
1 + + + +
3 + + + +
Copper (copper
sulphate)
5 + + – –
1 + + + +
3 + + + +
Chromium (potassium
chromate)
5 + – – +
1 + + + +
3 + + + +
Zinc (Zinc sulphate)
5 + – – +
1 + + + +
3 + + + +
Lead (Lead acetate)
5 + + + +
+  sensitive
–  resistant
Table 5. Screening for extra cellular enzyme production.
Bacterial isolates
Name of the enzyme
Proteus mirabilis Klebsiella pneumoniae Pseudomonas vesicularis Pseudomonas aeruginosa
Protease (Gelatin) + + + +
Protease (Casein) + + + +
Amylase (Starch) + + + +
Cellulase (Cellulose) – – – –
+  presence of activity
–  absence of activity
Table 6. Antibacterial activity of pure bacterial isolates.
Pathogens
Name of the isolates
Proteus mirabilis Klebsiella pneumoniae Micrococcus luteus Salmonella paratyphium
Proteus mirabilis – – + +
Klebsiella pneumoniae – – + +
Pseudomonas vesicularis – – + +
Pseudomonas aeruginosa + + + +
+  Presence of activity
–  Absence of activity

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588
Table 7. Antibacterial activity of isolates with organic solvent (Ethyl Acetate).
Pathogens
Name of the isolates
Proteus mirabilis Klebsiella pneumoniae Micrococcus luteus Salmonella paratyphium
Proteus mirabilis – – + +
Klebsiella pneumoniae + – + +
Pseudomonas vesicularis – – + +
Pseudomonas aeruginosa – + + +
+  Presence of activity
–  Absence of activity
Klebsiella pneumoniae, Proteus mirabilis and Salmo-
nella paratyphium. The same happened when the iso-
lates when dissolved in Ethyl acetate, an organic solvent.
But the presence of pathogens in drinking water is se-
rious health risks. Here the isolates like Proteus mirabilis,
was a causative agent of urinary tract infection and
Klebsiella pneumoniae was a causative of Pneumonia.
The Environmental officers in the local Government
employment owe it a duty to educate about the health
risk in taking this kind of contaminant sachet water and
to create awareness to the public when drinking water
standards are violated.
6. CONCLUSIONS
People are increasingly concerned about the safety of
their drinking water. As improvements in analytical
methods allow detecting impurities at very low concen-
trations in water, water supplies once consider pure are
found to have contaminants. The enforcement of the
regulation guiding water quality before the national
agency for Food and Drug Administration Control
(NAFDAC) to comply with the drinking water qualities
guidelines values as recommended by W.H.O. becomes
urgent.
7. ACKNOWLEDGEMENTS
We sincerely thank the Department of Biotechnology, Vels University,
to carryout this project.
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