World Journal of Microbiology & Biotechnology 14, 727730

Comparison of plate count agar and R2A

medium for enumeration of heterotrophic
bacteria in natural mineral water
Salvatore Massa,* Marisa Caruso, Francesca Trovatelli and Massimo Tosques
In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared
the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with
alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results
showed that counts with R2A medium/spread plates at 22 C and after a 7-day incubation period were more than
343% higher than those obtained with PCA/pour plate method. At 37 C and after a 3-day incubation period, the
R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium
spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should
be recommended for heterotrophic plate counts in natural mineral water.
Key words: Heterotrophic bacteria, plate count agar, R2A medium.

In Europe the procedure that has been stipulated for

the enumeration of heterotrophic bacteria in natural
mineral water is the pour plate method, which consists of
the use of Plate Count Agar (PCA) with an incubation
period of 72 h at 22 C and 24 h at 37 C. Numerous
investigations have, however, shown that this
standard method frequently gives lower counts of bacteria from drinking water than do other procedures and
media.
Soestbergen & Lee (1969) have emphasized the importance of oxygen tension in pour plates compared with
spread plates, while Klein & Wu (1974) found that heterotrophic microorganisms in water samples are susceptible to the transient stress of the warmed agar used
in the standard methods pour plate procedure, causing
signicantly decreased recoveries.

S. Massa and M. Tosques are with the Istituto di Produzioni e Preparazioni

Alimentari, Facolta di Agraria di Foggia, Via Napoli 25, 71100 Foggia; M. Caruso
is with the Dipartimento di Biologia, Difesa e Biotecnologia Agroforestali, Via N.
Sauro 85, 85100 Potenza; and F. Trovatelli is with the Dipartimento di Agrochimica e Agrobiologia, Universita della Tuscia, 01100 Viterbo, Italy. *Corresponding author.

Moreover, the composition of the media has also been

considered crucial. Less concentrated media, when used
for examination of fresh and drinking water, generally
yield considerably higher counts (Jones 1970; Dott 1980;
Means et al. 1981). Recently Reasoner & Geldreich (1979;
1985) have developed a medium, designated R2A, containing small amounts of the essential growth nutrient
(yeast extract, casamino acids, glucose and soluble
starch). In several studies this medium has been proved
to be more suitable than PCA in water distribution systems (Fiksdal et al. 1982; Maki et al. 1986; Gibbs & Hayes
1988).
Article 5 of the Directive (European Community 1980)
requires that natural mineral water should be characterized at source from a bacteriological point of view.
Furthermore, from a public health perspective it would
be desirable to know the types and numbers of bacteria
that are consumed with the consumption of natural mineral water. In order to recover as many viable bacteria
as possible, the purpose of this study was to compare
counts obtained with the European standard method
(PCA/pour plates) and counts with alternative test
methods (PCA/spread plates, R2A/pour plates and
R2A/spread plates).

1998 Kluwer Academic Publishers

World Journal of Microbiology & Biotechnology, Vol 14, 1998

727

S. Massa et al.
Table 1. Comparison between two mediaa (Plate Count Agar (PCA) and R2A medium) and two methods (pour plate and spread plate) in
counting heterotrophic bacteria after 3, 5 and 7 days incubation at 22 C.
Counts after

Medium/method
PCA/Pour plate
n = 122

PCA/Spread plate
n = 122

R2A/Pour plate
n = 122

R2A/Spread plate
n = 122

Rangeb
Mean (SD)c
Diff.d

022
0.89 (2.9)
0

048
1.12 (3.5)
+25.8

0106
3.07 (12.3)
+244.9

052
1.27 (3.8)
+42.7

5 days
Range
Mean (SD)
Diff.

034
1.77 (4.4)
0

054
1.60 (4.5)
)9.6

0111
4.46 (14.6)
+151.9

0158
5.53 (15.4)
+212.4

7 days
Range
Mean (SD)e
Diff.

0124
4.41 (13.7)
0

054
1.71x (5.7)
)61.2

0336
11.86 (23.8)
+168.9

01008
19.57y (63.4)
+343.7

3 days

According to Reasoner and Geldrich (1985);

Values are expressed as cfu ml)1;
c
Arithmetic mean (standard deviation);
d
Diff., differences of PCA/pour plate counts (example: + 25.8 = (1.12 ) 0.89 100/0.89);
e
Means in this row with different superscript differ signicantly (P < 0.05)
b

Materials and Methods

Sampling
A total of 112 natural mineral water samples were collected
between May 1993 and April 1994 from four bottling plants situated in the Basilicata area (Italy) at an altitude of nearly 300 m
above sea level. Sampling was done from the reservoir of the
bottling plant in sterile 1 l glass bottles. After collection, the
samples were transported to the laboratory, cooled on ice and
examined within 6 h.
Determination of colony forming units (CFU)
Two media were used to recover heterotrophic bacteria from
natural mineral water : PCA (Oxoid) and R2A medium. R2A
medium was prepared according to Reasoner & Geldreich
(1985) : yeast extract, 0.5 g; proteose peptone (Oxoid), 0.5 g; casamino acids (Difco), 0.5 g; glucose, 0.5 g; soluble starch, 0.5 g;
K2HPO4, 0.3 g; MgSO47H2O, 0.05 g; sodium pyruvate, 0.3 g;
agar, 15 g and demineralized water, 1000 ml (pH 7.2). Pour
plates in duplicate were inoculated with 1 ml of the water
samples and 1 ml of 10)1 and 10)2 dilutions prepared in sterile
mineral water of the same source. The molten agar media were
cooled to 45 C prior to pouring. Spread plates in duplicate were
inoculated with 0.1 ml of the samples and 0.1 ml of the 10)1 and
10)2 dilutions, and spread with a sterile glass rod. All plates
were incubated at 22 C for 3, 5 and 7 days and at 37 C for 1, 3
and 5 days.
Bacteria isolated at random from PCA and R2A medium
plates incubated at both 22 and 37 C were puried and classied with the schemes and methods used by LeChevallier et al.
(1980) and Chand et al. (1992). Gram-negative non-fermentative
rods were identied with API NE strips (Biomerieux, Marcy
l'Etoile, France) according to the instructions of the manufacturer.

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World Journal of Microbiology & Biotechnology, Vol 14, 1998

Statistical analysis
The least signicant differences (P < 0.05) of the mean counts
were calculated by Students' t-test (Cavalli-Sforza 1966). The
bacterial counts were converted into the log of the bacterial
counts+1. This conversion permitted the inclusion of zero
counts.

Results and Discussion

Tables 1 and 2 show the average counts obtained using
two temperatures of incubation (22 C for 3, 5 and 7 days
and 37 C for 1, 3 and 5 days), two media (PCA and
R2A), two plate count procedures (pour and spread
plates) and the percentage differences of the means of the
alternative test methods (PCA/spread plates, R2A/pour
plates and R2A/spread plates) with respect to the mean
of PCA/pour plates (standard method). Although a statistical signicant difference (P < 0.05; t-test) was found
only between the means of PCA/spread plate and R2A/
spread plate after seven-day counts (Table1), the difference of the means obtained at 22 C with R2A/spread
plate method were on average, more than 212 and 343%,
respectively after 5 and 7 days, higher than those obtained with the PCA/pour plate method. With R2A/
pour plate the differences of the mean counts were about
245% greater at 3 days, about 152% at 5 days, and about
169% at 7 days greater than the standard method. These
results are in accordance with those obtained by Gibbs &
Hayes (1988). When these authors processed an organi-

Enumeration medium for mineral water

a

Table 2. Comparison between two media (Plate Count Agar (PCA) and R2A medium ) and two methods (pour plate and spread plate) in
counting heterotrophic bacteria after 1, 3 and 5 days incubation at 37 C.
Counts after

Medium/method
PCA/Pour plate
n = 122

PCA/Spread plate
n = 122

R2A/Pour plate
n = 122

R2A/Spread plate
n = 122

Rangeb
Mean (SD)c
Diff.d

011
0.51 (1.9)
0

03
0.39 (1.8)
)23.5

027
0.94 (3.5)
+84.3

08
0.51 (2.5)
0

3 days
Range
Mean (SD)
Diff.

019
1.58 (3.2)
0

020
1.62 (5.3)
+2.5

0130
7.41 (24.2)
+368.9

075
3.68 (12.0)
+132.9

5 days
Range
Mean (SD)
Diff.

068
3.71 (25.7)
0

080
4.0 (12.6)
+7.8

0152
11.64 (34.6)
+213.7

0126
9.99 (48.7)
+185.7

1 day

According to Reasoner and Geldreich (1985);

Values are expressed as cfu ml)1;
c
Arithmetic mean (standard deviation);
d
Diff., differences of PCA/pour plate counts (see Table 1 for explanation).
b

Table 3. Summary of bacteria isolated from PCAa and R2A mediumb.

22 C
PCA
Pseudomonas uorescens
Pseudomonas vesicularis
Pseudomonas stutzeri
Fluorescent pseudomonads (not identied)
Comamonas acidovorans
Acinetobacter junii
Flavobacterium spp.
Arthrobacter spp.
Total
a
b
c

8 (24.2)c
5 (15.1)
3 (9.0)
15 (45.4)
1 (3.0)
1 (3.0)
0
0
33 (100)

37 C
R2A

PCA

R2A

7 (18.4)
1 (2.6)
2 (5.2)
12 (31.5)
1 (2.6)
0
10 (26.3)
5 (13.1)
38 (100)

4 (11.4)
6 (17.1)
0
22 (62.8)
1 (2.8)
2 (5.7)
0
0
35 (100)

5 (13.8)
4 (11.1)
0
16 (44.4)
0
1 (2.7)
7 (19.4)
3 (8.3)
36 (100)

PCA, Plate Count Agar;

According to Reasoner and Geldreich (1985);
Number of strains (%).

cally rich treated water (total organic carbon 5 mg l)1),

they found that the use of R2A/spread plate gave average counts 39 times greater at 3 days and 27 times greater
at 7 days than the Yeast Extract Agar medium (which is
similar to PCA); furthermore, counts obtained using R2A
medium and the pour plate technique were on average 4
and 8 times greater than counts using the YEA medium
and pour plate techniques. Gibbs & Hayes (1988) also
found, in contrast to our results, that the YEA/spread
method gave average counts seven times greater than the
pour plate method at both 5 and 7 days. Our results
illustrate clearly that for an incubation temperaturean of
22 C, the R2A/surface plate method is better than the
PCA/pour plate method for determining the heterotrophic bacteria in natural mineral water.

At 37 C the mean counts did not differ signicantly

(P > 0.05; t-test). However, the counts obtained on the
third day with R2A medium were higher than with PCA
(Table 2). In particular with the R2A/pour method, the
counts were more than 368% those obtained with the
standard method after 3 days of incubation. Although the
counts on R2A/spread plates increased from the third to
the fth day, they remained lower than those obtained
with R2A/pour plates. As observation stopped on the
fth day, we are unable to predict whether subsequent to
longer incubation the counts on R2A/spread plates
would have remained lower, been equal to, or even
higher than those obtained with R2A/pour plates. It is, in
fact, well known that in general when the medium is the
same, the counts obtained with the spread plate proce-

World Journal of Microbiology & Biotechnology, Vol 14, 1998

729

S. Massa et al.
dure are higher than those obtained with the pour
plate method because of the stress inicted on the microorganisms when they come into contact with the
molten agar at a temperature of 45 C (Klein & Wu 1974).
Table 3 contains a summary of bacteria tentatively
identied from PCA and R2A medium. Pseudomonas,
Comamonas and Acinetobacter species were isolated, irrespective of temperature, both from PCA and R2A medium. Flavobacterium spp. and Arthrobacter spp. were
isolated only from R2A medium, indicating that with this
substrate it is possible to recover a greater diversity of
bacterial genera. While Flavobacterium species may be
opportunistic pathogens where there are populations of
compromised individuals (von Graevenitz 1985),
Arthrobacter species are able to grow at low nutrient levels and were reportedly responsible for water colour
and taste problems (LeChevallier et al. 1987). Other authors have found that complex minimal media (e.g. R2A)
lead to recovery of more strains from different bacterial
genera than the commonly used PCA (Dott 1980; Means
et al. 1981).
In conclusion, the results show that R2A medium may
be more suitable when compared with PCA in agreement
with other investigators (Fiksdal et al. 1982; Maki et al.
1986; Gibbs & Hayes 1988), who have employed this
medium for other types of water. R2A medium, which
has not hithero been generally used for bottled water,
should be recommended in order to recover as many
viable bacteria as possible and to help the clarication of
any public health effects associated with heterotrophic
bacteria present in natural mineral water.

Acknowledgement
The authors are indebted to Dr. Marzia Albenzio for
technical assistance.

References
Cavalli-Sforza, L. 1996. Analisi statistica per medici e biologi. Torino
Italy: Editore Boringhieri.
Chand, T., Harris, R.F. & Andrew, J.H. 1992 Enumeration and
characterization of bacterial colonist of a submersed acquatic
plant, eurasian watermil foil. (Myriophillum spicatum L.).
Applied and Environmental Microbiology 58, 33743379.
Dott, W. 1980 Qualitative and quantitative Bestimmung von
Bakterienpopulationen aus aquatischen Biotopen, 1. Mittei-

730

World Journal of Microbiology & Biotechnology, Vol 14, 1998

lung: Selektionierung von Bakteriengruppen durch Isolierung

auf verschiedenen Nahrboden. Zentralblatt fur Hygiene, 1.
Abteilung Originale B 170, 9398.
European Community 1980 Council Directive No. 80/777/EEC
of 15 July 1980 on the approximation of the laws of the
member states relating to the exploitation and marketing of
natural mineral waters. Ofcial Journal of the European Communities L 229, 110.
Fiksdal, L., Vik, E.A., Mills, A. & Staley, J. T. 1982 Non standardmethods for enumerating bacteria in drinking water. Journal
of American Water Works Association 74, 313318.
Gibbs, R.A. & Hayes, C.R. 1988 The use of R2A medium and the
spread plate method for the enumeration of heterotrophic
bacteria in drinking water. Letters in Applied Microbiology 6,
1921.
Jones, J.G. 1970 Studies on freshwater bacteria: effect of medium
composition and method on estimates of bacteria populations. Journal of Applied Bacteriology 33, 679686.
Klein, D.A. & WU, S. 1974 Stress: a factor to be considered in
heterotrophic microorganisms enumeration from aquatic
environments. Applied Microbiology 27, 429431.
LeChevallier, M.W., Babcock, T.M. & Lee, R.G. 1987 Examination and characterization of distribution system biolms.
Applied and Environmental Microbiology 53, 27142724.
LeChevallier, M.W., Seidler, R.J. & Evans, T.M. 1980 Enumeration and characterization of standard plate count bacteria in
raw and chlorinated water supplies. Applied and Environmental Microbiology 40, 922930.
Maki, J.S., Lacroix, S.J., Hopkins, B.S. & Staley, J.T. 1986
Recovery and diversity of heterotrophic bacteria from
chlorinated drinking water. Applied and Enviromental Microbiology 51, 10471055.
Means, E.G., Hanami, L., Ridgeway, H.F. & Olson, B.H. 1981
Evaluating mediums and plating techniques for enumerating
bacteria in water distribution systems. Journal of American
Water Works Association 73 , 585590.
Reasoner, D.J. & Geldreich, E.E. 1979 Abstracts of Annual Meeting
of American Society of Microbiology 7, 180.
Reasoner, D.J. & Geldreich, E.E. 1985 A new medium for the
enumeration and subculture of bacteria from potable water.
Applied and Environmental Microbiology 49, 17.
Soestbergen, A.A. van & Lee, C.H. 1969 Pour plates or streak
plates? Applied Microbiology 18, 10921093.
von Graevenitz, A. 1985 Ecology, clinical signicance, and
antimicrobial susceptibility of frequently encountered glucose
non-fermentative gram-negative rods, pp. 181232. In Nonfermentative, Gram-negative Rods: Laboratory Identication and
Clinical Aspect., ed Gilardi. G.L., New York: Marcel Dekker,
Inc., ISBN 0-8247-7370-5.

(Received in revised form 18 February 1998;

accepted 19 February 1998)