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International Journal of Food Microbiology 48 (1999) 5965

A survey of the microbiological quality of bottled water sold in the

UK and changes occurring during storage
A. Benito Armas, J.P. Sutherland
Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 6BZ, UK
Received 23 June 1998; received in revised form 17 December 1998; accepted 10 February 1999
Eight brands of domestic and imported bottled water were microbiologically analysed within three hours of purchase at a
local supermarket. Viable numbers of microorganisms were estimated on Plate Count Agar (PCA) and PCA diluted to quarter
and tenth strengths (1/ 4 PCA and 1/ 10 PCA) and incubated at temperatures of 10, 15, 25 and 378C. Plate count agar diluted
4 21
to 1/ 4 and 1/ 10 incubated at 258C yielded the highest initial counts, up to 10 cfu ml . Pseudomonas spp. was the
predominant species. After 6 months of storage at room temperature (18258C), few quantitative and qualitative differences
were found in the microora. 1999 Elsevier Science B.V. All rights reserved.
Keywords: Bottled water; Microbial growth; Storage
1. Introduction about tap water, since bottled water is often regarded
as safer and healthier than tap water.
During the past decade, there has been a consider- According to the European Community Directive
able increase in the consumption of bottled water in (1980), which became law in the UK under the
the UK, and this trend is expected to continue. Natural Mineral Water Regulations (Ministry of
Moreover, uncarbonated water is now considerably Agriculture, Fisheries and Food, 1985), natural min-
more popular than carbonated, having become a eral water is not sterilised, pasteurised or otherwise
substitute for tap water in some households. (Anon, treated to remove or destroy microorganisms. The
1991, 1994, 1995). This reects consumer concerns number of bacteria recovered at the source is gener-
ally very low, around 10 cfu ml , but there are
many reports that viable counts increase, notably in
4 5 21
uncarbonated water, to 10 10 cfu ml after 13
*Corresponding author. Present address: School of Health and
weeks of storage (Schmidt-Lorenz, 1976; Gonzalez
Sports Science, University of North London, 166-220 Holloway
et al., 1987; Bischofberger et al., 1990; Mavridou,
Road, London N7 8DB. Tel.: 144-171-753-7023; fax: 144-171-
1992; Mavridou et al., 1994; Tsai and Yu, 1997).
E-mail address: (J.P. Sutherland) Those organisms were mainly Gram negative non-
0168-1605/ 99/ $ see front matter 1999 Elsevier Science B.V. All rights reserved.
PI I : S0168- 1605( 99) 00027- 6
60 A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965
fermentative rods, frequently identied as Pseudo- chloride (PVC) bottles (Table 1) were purchased
monas spp. and related genera, originating from the from a local supermarket.
water itself (autochthonous microora) or contami-
nants introduced during the bottling process (alloch- 2.2. Sampling
thonous microora). The public health signicance
of the high numbers of Pseudomonas spp. which One bottle of each type of water was sampled
develop is unclear (Hunter, 1993). Many Pseudo- immediately after purchase and a second was sam-
monas spp. recovered from water are resistant to pled after storage for six months in shaded daylight
antimicrobial agents (Hernandez Duquino and at ambient temperature (18258C) in the laboratory.
Rosenberg, 1987). Microbial numbers were estimated by decimal
Bottled water has been the vehicle of transmission dilution in 1/ 4 strength Ringers solution (Oxoid
of Vibrio cholerae, causing an outbreak of cholera in BR52) and plating on eight plates of each of the
Portugal (Blake et al., 1977) and historically, re- following media: Plate Count Agar (PCA; Oxoid
covery of Staphylococcus aureus (Leclerc et al., CM325) at full strength, quarter-strength (1/ 4 PCA)
1985) and Aeromonas hydrophila (Gonzalez et al., and tenth-strength (1/ 10 PCA). Duplicate plates of
1987; Manaia et al., 1990) from bottled water have each of the three agars were incubated at four
caused concerns about its safety. temperatures: 10, 15, 25, and 378C. Plates were
The purpose of this study was to evaluate the counted after 214 days of incubation, depending on
quantitative and qualitative microbiological status of temperature of incubation.
indigenous and imported bottled water sold in the If no colonies were recovered with the surface
UK and to determine any changes occurring during plating technique, 1 ml pour plates were prepared
storage at room temperature, with the aim of iden- with the same media, taking 1 ml from the same
tifying optimal recovery conditions for the micro- bottle as the original sample.
ora, with respect to media and temperature of
incubation. 2.3. Statistical evaluation
The following null hypotheses were examined: (a)
There was no signicant difference between the
2. Materials and methods numbers of microorganisms recovered from the
different brands of bottled water; (b) There was no
2.1. Bottled water signicant difference between the numbers of micro-
organisms initially present in the water and those
Three bottles, all with the same expiry date, of present after storage for 6 months at ambient tem-
each of eight brands of domestically-produced and perature; (c) There was no signicant difference in
imported non-carbonated bottled water in polyvinyl the numbers of microorganism recovered at tempera-
Table 1
Analysis of eight brands of bottled water
a b b b b b b b b
Brand Origin pH Na K Ca Mg Cl SO NO HCO
4 3 3
d c
A UK NR 10 3 102 24 30 60 6 NA
B Belgium 5.8 3 0.5 3.5 1.3 5 6.5 1.9 11
C France NR 8 5.4 10.4 6 7.5 6.7 4 64
D UK 7.4 24 1 55 19 42 23 NA 248
E France 7.2 5 1 78 24 4.5 10 3.8 357
F France NR 12 3.2 100 12 21 12 0.6 275
G UK 7.6 9 1 39 15 15 7 1 190
H UK 7.6 65 1 48 13 60 12 3.5 180
Measured in laboratory.
b 21
Information on label of bottle mg l .
NA data not available.
NR data not recorded.
A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965 61
tures of 10, 15, 25 and 378C; (d) There was no were used for further identication of microorga-
signicant difference in recovery attributable to the nisms: API-20NE (Biomerieux, Basingstoke, UK)
concentration of PCA used for recovery. for Gram-negative, non-fermentative organisms and
To test these hypotheses, analysis of variance API-STAPH for Gram-positive cocci. Additional
(ANOVA) of the microbiological data was carried tests, including lecithinase production, hydrolysis of
out by the maximum likelihood estimation in SAS Tween 80, growth in 4% NaCl, growth on MacCon-
Proc Lifereg. key agar (Oxoid CM115), growth at 428C, fermen-
tation of sorbitol, acid production from xylose and
2.4. Bacterial identication motility (Gilardi, 1971) were necessary in some
cases for nal identication.
Randomly-selected colonies from each plate were
checked for purity by streaking on fresh agar
medium of the same strength as that from which they
3. Results and discussion
were isolated. The streaked plate was incubated at
the same temperature as that on which the original
3.1. Analytical characteristics of waters
count was estimated.
Isolates thus puried were initially screened by
The analytical characteristics of the different
Gram stain, cytochrome oxidase test, catalase test
waters are shown in Table 1.
and the ability to oxidise or ferment glucose (Hugh
and Leifson, 1953; Gerhardt et al., 1981). Other
microbial characteristics recorded included: colonial 3.2. Bacterial numbers
morphology, production of water-diffusible pigment,
ability to grow at low temperatures and, for those Table 2 shows viable counts (cfu ml ) in the
organisms isolated on lower-strength media, ability eight brands of mineral water sampled on the day of
to grow on full-strength agar. The following systems purchase and after 6 months of storage, with various
Table 2
Heterotrophic plate count (cfu ml ) in eight brands of uncarbonated bottled water on day of purchase (count 1) and after 6 months storage
(count 2) using different media and incubation temperatures
Brand 108C 158C 258C 378C
PCA 1/ 4PCA 1/ 10PCA PCA 1/ 4PCA 1/ 10PCA PCA 1/ 4PCA 1/ 10PCA PCA 1/ 4PCA 1/ 10PCA
21 21 21 21 21 21 21 21 21 21 21 21
cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml cfu ml
b 3 4 4
A1 ** 7.7 310 ** ** 2.4 310 ** ** 2.4 310 ** ** ** **
3 3 3 3 3 3 2 3 2
A2 1.7 310 1.0 310 1.5 310 1.5 310 2.0 310 1.2 310 ** 7.5 310 1.5 310 ** 7.5 310 **
3 3 4 3 3
B1 ** ** 8.0 310 ** 8.0 310 1.6 310 ** 3.6 310 4.7 310 ** ** **
2 2 2 3 3
B2 ** ** 5.0 310 ** ** 5.0 310 5.0 310 1.0 310 1.0 310 ** ** **
4 4 4 4 4 4 4 4 4
C1 3.5 310 3.3 310 3.9 310 4.4 310 3.4 310 5.0 310 2.9 310 5.8 310 5.2 310 ** ** **
3 3 3 2 3 3 3 3 3
C2 1.0 310 1.5 310 1.2 310 5.0 310 1.8 310 1.7 310 2.2 310 8.7 310 8.1 310 ** ** **
3 3 3 3 3 3 3 3 3
D1 2.0 310 4.8 310 4.6 310 2.2 310 3.7 310 4.2 310 4.8 310 4.8 310 7.2 310 ** ** **
3 3 3 3 3
D2 ** ** ** ** 2.7 310 3.5 310 3.0 310 2.0 310 1.0 310 ** ** **
3 3 3 3 3 3 3 3 3 3 2 2
E1 4.0 310 3.6 310 4.5 310 4.7 310 4.2 310 4.2 310 3.0 310 4.6 310 5.6 310 1.5 310 5.0 310 5.0 310
2 2 3 3 3 3
E2 ** ** ** 7.5 310 5.0 310 1.7 310 2.0 310 1.5 310 2.5 310 ** ** **
F1 * * * * * * * * * * * *
F2 ** ** ** ** ** ** ** ** ** ** ** **
4 4 4 4 4 4 4 4
G1 ** 2.6 310 2.8 310 1.2 310 3.1 310 3.2 310 2.3 310 2.2 310 2.5 310 ** ** **
3 3 3 4 4 3 4 4
G2 ** 4.5 310 4.7 310 4.6 310 2.5 310 2.6 310 7.5 310 4.5 310 4.6 310 ** ** **
3 3 3 3 4 4 4 4 4
H1 7.3 310 7.5 310 7.5 310 6.8 310 1.3 310 2.1 310 2.2 310 3.7 310 5.0 310 ** ** **
3 4 4 3 4 4 4 4 4
H2 6.4 310 1.4 310 1.4 310 7.6 310 1.9 310 2.1 310 1.0 310 2.3 310 2.4 310 ** ** **
AH represent different brands of water, sufxes 1 and 2 represent respectively numbers obtained in the initial sample and after 6
months of storage.
b 21
** ,500 cfu ml .
c 21
* ,1 cfu ml .
62 A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965
strengths of PCA and different incubation tempera- covered from water of this type, which is low in
tures. nutrients and not heat-treated, absence of micro-
4 21
Initial microbial numbers of up to 10 cfu ml organisms may suggest the presence of toxic sub-
were recovered at temperatures of 10, 15 and 258C stances in the water (Geldreich and Clark, 1965;
from most of the bottles of water examined, on all Manaia et al., 1990).
concentrations of PCA. At 378C, microbial numbers
were generally ,500 cfu ml , with the exception
2 21
of E1 (5.0 310 cfu ml on both reduced strength 3.3. Statistical evaluation
3 21
agars and 1.5 310 cfu ml on full strength PCA).
After 6 months storage, there appeared to have Table 3 shows the outcome of the analysis of
been an overall decrease in microbial numbers (apart variance applied to brands A, B, C, D, E, G and H at
from A, which showed a slight increase) recovered at temperatures of 10, 15 and 258C to test the null
the three lower incubation temperatures (Table 2). At hypotheses. Brand F was omitted from the analysis
2 21
378C, a count of 7.5 310 cfu ml on quarter- and there were insufcient data from any of the
strength PCA was obtained from sample A2, while water samples at 378C for inclusion.
the organisms originally recovered from sample E1 The rst null hypothesis, that there was no signi-
had declined in number to ,500 cfu ml in sample cant difference between the numbers of microorga-
E2. nisms recovered from the different brands of bottled
Some brands of bottled water (C1, D1, E1 and water, is not true since there were signicant differ-
H1) yielded similar counts at temperatures 10258C ences in microbial numbers between the brands of
on the three strengths of PCA used to recover the water. This was also observed by Tsai and Yu (1997)
organisms; but in others, bacteria failed to grow on who recorded heterotrophic plate counts ranging
2 21 5 21
full strength media (A1, B1 and G1), especially at from ,10 cfu ml to .10 cfu ml from a
lower temperatures. This suggests inhibition of or- selection of domestic and imported bottled waters in
ganisms in these brands by high concentrations of Taiwan, and Bischofberger et al. (1990) who iden-
nutrients in the recovery medium as observed by tied qualitative and quantitative differences between
others (Schmidt-Lorenz, 1976; Schwaller and bottled water from two springs.
Schmidt-Lorenz, 1980). One initial bottle and one bottle of water stored for
In brand F, a French import, no bacteria were 6 months for each brand were examined, which may
recovered initially or after storage for 6 months, even appear to limit the reliability of the conclusions that
from pour plates prepared with 1 ml of undiluted can be drawn from the results of the rst null
water in the three different strengths of agar. Since it hypothesis. However, this was a small scale pre-
is anticipated that bacteria would normally be re- liminary experiment with the aim of developing the
Table 3
Analysis of variance of bottled waters.
Term Degrees of Chi-squared Signicance
intercept 1 741.3 0.0001
brand of water 6 115.5 0.0001
age (new or 6 months) 1 2.3 NS
strength (of PCA) 2 6 0.0507
temperature (of incubation) 2 0.4 NS
age 3brand 6 100.9 0.0001
temperature 3strength 4 3.8 NS
age 3strength 2 2.7 NS
age 3temperature 2 12 0.0025
brand 3strength 12 89.9 0.0001
brand 3temperature 12 18.7 0.096
NS: not signicant.
A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965 63
methodology to be employed in a future, more undiluted or tenth-strength PCA, while for brand B,
comprehensive, study. undiluted PCA gives a signicantly lower recovery
Since there was no signicant difference in micro- than quarter-strength, which in turn was signicantly
bial numbers between the primary and six month lower than tenth-strength PCA. For brands C, G and
samples, it would initially appear that the second null H, undiluted PCA gave lower recoveries than quar-
hypothesis is true, as there was no general trend for ter- and tenth-strength agar, but the latter did not
the numbers in any brand of water to be higher at differ signicantly. For brands D and E there was no
one of the two time points (age effect, Table 3). signicant effect of the dilution of the agar.
However, there was a signicant interaction between
age and brand of water (Table 3), indicating that for 3.4. Preliminary differentiation of isolated bacteria
some brands of bottled water there was a signicant
difference in microbial numbers attributable to stor- After screening more than 700 isolates from the
age time. Further analyses revealed that brands B, C, initial sampling and a further 300 after 6 months
D and E had signicantly lower counts after 6 storage, a considerable predominance of Gram nega-
months storage, while for brands A, G and H there tive over Gram positive bacteria was evident, with
was no signicant change with storage time. Thus it percentages of Gram negatives of 97.5% and 93.4%
appears that microorganisms in the different brands in the rst and second sampling respectively, con-
of water are affected differently by ageing. Numbers rming other reports (Quevedo-Sarmiento et al.,
of microorganisms did not increase signicantly in 1986; Hunter and Burge, 1987; Mavridou, 1992;
any sample during storage. Mavridou et al., 1994; Tsai and Yu, 1997). Gram
The third null hypothesis, that there is no signi- negative oxidative bacteria, possessing cytochrome
cant difference between recovery of organisms at oxidase and catalase, constituted more than 50% of
temperatures of 10, 15 and 258C is also apparently the isolates on both occasions of sampling. Gram
true (incubation temperature effect, Table 3). Never- negative fermentative organisms were not identied
theless, there is a signicant interaction between age at any stage. Gram positive, catalase positive, fer-
and incubation temperature for fresh samples, since mentative bacteria were found occasionally in some
recovery at 108C was signicantly lower than at 15 brands of water.
or 258C. In the samples stored for 6 months, this
effect became more pronounced, since there was a 3.5. Identication of selected bacteria with API
signicant interaction between time of storage and systems
incubation temperature. The numbers recovered at
108C were signicantly lower than at 158C which in The results of identication of some isolates with
turn were signicantly lower than those at 258C. This API-20NE and API-STAPH systems are shown in
suggests that there may have been a change in the Table 4. Pseudomonas was the predominant genus
composition of the microora to a population whose identied with API-20NE and was recovered from
growth is favoured by the ambient storage tempera- all waters tested except brand F, from which no
ture. organisms were isolated. Ps. uorescens and Ps.
The fourth null hypothesis, that there is no signi- vesicularis were the most frequently isolated species
cant effect of agar strength on numbers of micro- (Table 4). Mavridou et al. (1994) recorded Ps.
organisms recovered, is not true. There is a marginal- uorescens and Ps. stutzeri as the most commonly
ly signicant effect of agar strength (Table 3), isolated organisms from Greek bottled water. Hunter
indicating that the agar strength affects recovery (1993) recovered predominantly Ps. stutzeri and Ps.
from all brands of water under all conditions. Further putida from bottled waters from Germany and
analysis showed that recovery on undiluted PCA is America, with Portuguese water having an abnormal-
signicantly lower than on either of the more dilute ly high proportion (45%) of Ps. aeruginosa. Morais
agars, which are not signicantly different. This and Da Costa (1990) recovered a large proportion of
conrms the observation made above. The brand of Pseudomonas spp. from a single type of Portuguese
water also has an effect to the extent that, for brand water, but with the exception of Ps. vesicularis, none
A, quarter-strength PCA gave a higher recovery than could be speciated with certainty. Bischofberger et
64 A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965
Table 4
Percentage of identied isolates from different brands of mineral water isolated on day of purchase (sample 1) and after 6 months storage
(sample 2)
A1 A2 B1 B2 C1 C2 D1 D2 E1 E2 G1 G2 H1 H2
Pseudomonas uorescens 14.3 100 33.3 73 8.7 26.1 66.6 9.1
vesicularis 71.4 77.8 16.7 40 4.2 23.5 17.4 42.8
paucimobilis 16.7 5.9 7.1
diminuta 33.3 40 8.3 5.9 36.4
mesophilica 8.7 7.1
Burkholderia cepacia 8.3
Sphaeromonas paucimobilis 4.3
Flavobacterium oryzahabitans 5.9 4.3
Comamonas sp. 14.3 13
Acinetobacter junii 6.6
CDCgr.IVC-2d 8.3 28.6 4.3 7.1
Moraxella 4.2
Staphylococcus spp. 16.7 4.2 8.7 8.7
other Gram negative 14.3 22.2 33.3 20 12.5 27 57.1 69.6 58.8 26.2 35.9 26.8 54.5
non-fermentative rods
al. (1990) observed that the species of Pseudomonas method for examination of bottled water to obtain
were characteristic of the source of water. total viable counts with only diluted media and
A large number of isolates could not be identied incubation at 258C, conditions that yield the highest
with the API system due to the limitations of the counts in these experiments.
database. Although the API 20NE system has been Different opinions have been expressed about the
used to identify bacteria from bottled water (Manaia public health signicance of Pseudomonas and re-
et al., 1990; Mavridou, 1992), it is often unsatisfac- lated species in bottled water. They are reported to
tory for identication of bacteria from this environ- be resistant to several antimicrobial agents (Her-
ment, having been developed specically for identi- nandez Duquino and Rosenberg, 1987) and strains of
cation of bacteria of clinical origin. Additional tests Burkholderia (Pseudomonas) cepacia (isolated from
were therefore necessary in most cases for discrimi- the second sample of brand C; Table 4) have been
nation of species. associated with infections in immunocompromised
The types of bacteria recovered can depend on the individuals and are sometimes a cause of chest
culture techniques used. The pour-plate technique infections in children with cystic brosis (Hunter,
(the ofcial method of the European Community 1993). However, other studies have shown a minimal
Directive, 1980) with incubation at temperatures of risk of infection from drinking these waters (Leclerc,
228C and 378C tends to favour recovery of fast- 1980).
growing fastidious mesophilic and psychrotrophic Gram positive cocci were detected in waters B, C,
bacteria. To ensure that all the viable bacteria in E, and G. The presence of Gram positive cocci in
bottled water are recovered, surface culture tech- bottled water has also been reported by Hunter and
niques with low-nutrient media and longer incuba- Burge (1987) and Mavridou (1992). Their signi-
tion at lower temperatures are advisable (Bischof- cance is unclear, although Hunter and Burge (1987)
berger et al., 1990). and Hunter (1993) concluded that since the human
Our results, however, showed that although higher commensals Staph. epidermidis and Staph. hominis
recoveries were obtained on more dilute media, there were among the Gram positive cocci isolated from
were no differences between the composition of the the waters, they were probably contaminated by
microora isolated at different temperatures, and people at the stage of lling and are thus part of the
bacteria isolated on full strength PCA were generally allochthonous microora.
similar in identity to those isolated on 1/ 4 and 1/ 10 It was not possible to determine in this study
PCA. From these results, it is possible to simplify the whether the microora was autochthonous or alloch-
A.B. Armas, J.P. Sutherland / International Journal of Food Microbiology 48 (1999) 5965 65
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