Food Research International, Vol. 30, No. 314, pp.

207-211, 1997
% 1997 Canadian Institute of Food Science and Technology

Published by Elsevier Science Ltd

Printed in Great Britain

ELSEVIER

PII:

SO963-9969(97)00044-6

0963-9969/97 $17.00 + 0.00

Comparison of ten media for the enumeration of

yeasts in dairy products
J. J. Welthagen & B. C. Viljoen
Department

qf Food Science, PO Box 339, U.O.F.S., Bloemfontein, 9300, South Africa

Ten selective mycological media were evaluated for their suitability to enumerate
yeasts in six different dairy products. Although variability was observed in counts
obtained for individual dairy products, no significant overall differences
(p > 0.05) were observed among the 10 selective plating media. Antibiotic-supplemented media such as oxytetracycline glucose yeast agar (OGY), yeast extract
glucose chloramphenicol agar (YGC), rose bengal chloramphenicol agar (RBCA)
and rose bengal chlortetracycline agar (RBC) are superior to acidified potato
dextrose agar and other acidified media for yeast enumeration in dairy products
with neutral pH values. No significant differences (p > 0.05) among the 10

selective media ability to enumerate yeasts in dairy products at low pH values

were observed. 0 1997 Canadian Institute of Food Science and Technology.
Published by Elsevier Science Ltd
Keywords: selective media, yeasts, dairy products, enumeration.

(1950) noticed the inhibitory effect of rose bengal on the

mould colony growth of soil fungi. Overcast and
Weakly (1969) confirmed these findings for moulds isolated from cottage cheese. Jarvis (1973) compared a
modification of the medium of Overcast and Weakly
(1969), which contains a combination of rose bengal and
chlortetracycline, versus oxytetracycline glucose yeast
agar medium. Both media suppressed bacterial growth
but only the rose bengal medium had a limited effect on
the growth of the mould colonies. The ideal medium for
enumeration of yeasts should suppress growth of bacteria and moulds, and support growth of all yeasts present
(Beuchat, 1993). Although this ideal medium does not
exist, several satisfactory media are available and most
yeasts of interest to the dairy industry are enumerated by
standard methods (Fleet and Mian, 1987; Baroiller and
Schmidt, 1990; Rohm et al., 1992; Beuchat, 1993).
Media typically used are potato dextrose agar (Baroiller and Schmidt, 1990), malt extract agar (Fleet and
Mian, 1987), yeast extract glucose agar (Nooitgedagt
and Hartog, 1988; Rohm et al., 1992), yeast nitrogen
base (Besancon et al., 1992) and similar media supplemented with various antibiotics like oxytetracycline,
chlortetracycline,
chloramphenicol,
gentamycin and
streptomycin (Beuchat, 1993). Often a combination of
two antibiotics at levels in the order of l&lOOpg ml-

INTRODUCTION
Antibiotic and acidified amended media are currently
recommended for use in enumerating yeasts and moulds
in foods (APHA, 1976, 1978). Acidified media have
been used for many years for isolating fungi from dairy
products, soils and foods with the H-ion concentration
limiting growth of bacteria (Koburger, 1971; Nelson,
1972; Koburger and Farhat, 1975; Beuchat, 1979). The
disadvantage of acidified amended media is the inability
of stressed fungi to tolerate the low pH of the media
(Henson et al., 1982). Several workers have noted advantages in the use of media at pH 7.0 containing antibiotics or other antibacterial agents (Martin, 1950; Mossel
et al.,
1962; Overcast and Weakly, 1969; Fleet and
Mian, 1987; Beuchat, 1993). Various other attempts
have been made to improve fungal enumeration media.
Smith and Dawson (1944) discovered an inhibitory effect
of rose bengal on the growth of fungi isolated from soil.
A combination of this dye with streptomycin or chlortetracycline showed that none of the combinations prepared appeared to be fully effective in controlling
bacterial growth (Martin, 1950; Cooke, 1954). Martin
*To whom correspondence should be addressed. Fax: 27514480692; e-mail: Bennie@Landbou.UOVS.AC.ZA
207

J. J. Welthagen, B. C. Viljoen

208

are used to effectively inhibit bacterial growth (Beuchat,

1993).
For the isolation of yeasts from dairy products, there
is still a need for a selective medium to ensure reliable
quantitative enumeration of yeast colonies. In this
paper, we compare 10 different media for the isolation
of yeast colonies from six different dairy products. The
aim of this study was to select the most suitable medium
for the isolation of yeasts from dairy products.

MATERIALS

AND METHODS

Samples
Ten lots each of six different dairy products were purchased from a range of local supermarkets and immediately analyzed for microbial status. Within a given
dairy product, the ten samples in each lot, represented
samples from the same brand. The dairy samples included Cheddar cheese, raw milk (Dairy Belle Foods,
Bloemfontein, South Africa), yoghurt, milk powder,
cottage cheese and butter.
Media
Acidified potato dextrose agar (PDA-A; Oxoid CM
139) was prepared according to the manufacturers
directions and adjusted to a final pH of 3.5 f 0.1 with
sterile 10% tartaric acid. The modified rose bengalchlortetracycline agar (RBC; Oxoid CM 549), with a
final pH of 7.2, was prepared as described by Jarvis
(1973). The oxytetracycline-glucose-yeast
agar (OGY)
was prepared according to the manufacturers directions
(Merck 10877) and adjusted to a final pH of 6.6. In
order to temper OGY medium, 0.1 g-l (filter sterilized)
oxytetracycline
was added aseptically. Rose-bengal
chloramphenicol agar base (Oxoid CM 549) and yeast
extract glucose chloramphenicol (YGC; Merck 16000)
agar was prepared according to the manufacturers
directions. The final pH of the YGC medium was 6.6.
The standard yeast-malt extract (YM) agar was prepared
as described by Wickerham (1951). A second YM-based
medium was prepared with the substitution of glucose
by lactose. The final pH was adjusted to 3.5 for both
YM-based media with sterile 10% tartaric acid solution.
YM agar was also prepared as described by Wickerham
(1951), but with the addition of chloramphenicol selective supplement (Oxoid SR078E) to suppress the growth
of bacteria. The YM agar with glucose as carbon source
and chloramphenicol (pH 3.5), was enriched with 5%
(v/v) ethanol as an additional carbon source.
Examination of dairy food samples
Ten gram samples of each dairy product were homogenized in sterile plastic bags with the aid of a Colworth

400 stomacher

(Stomacher 400 Lab-Blender, Seward

Medical UAC House, London) for 2 min in 90 ml sterile
peptone water. All samples were performed in triplicate.
Further dilutions were prepared as necessary in 9ml
sterile peptone water. Samples were spread plated and
the plates incubated at 23 f 2C and counted after
5 days (APHA, 1978).
Statistical
All results were tabulated and statistically analyzed
employing the Analysis of Variance (ANOVA). Means
were compared by using the Student-Neumann-Keuls
test. A significant F-value of p f 0.05 was employed
(Schefler, 1979).

RESULTS

AND DISCUSSION

The mean yeast counts with the standard error present

in the six dairy products, using 10 different selective
media and 10 samples within each product, are shown
in Table 1. Results were analyzed with all possible
interactions examined. The 10 different samples of the
same brand within each dairy product were separately
analysed for each of the 10 media in triplicate (each
medium x 10 samples of each product x triplicate)
No yeast counts were obtained during this study on
any of the 10 selective media inoculated with samples of
milk powder, while relatively low yeast counts were
encountered for butter, cottage cheese and Cheddar
cheese. The absence of yeasts in milk powder is in
agreement with the literature (Desk, 1991; Beuchat,
1993) indicating that the growth of yeasts is limited due
to the low aw (Fleet, 1990). Previous studies, though,
have shown that yeasts can spoil cottage cheese or
similar types of unripened cheeses such as quarg (Roth
et al., 1971; Hankin et al., 1975; Guirand and Galzy,
1976; Brocklehurst and Lund, 1985; Engel, 1986; Engel
et al., 1987). Yeast counts of up to lo2 cells g-i were
obtained from the cottage cheese while no significant
overall differences (p > 0.05) among any of the ten
media in their ability to enumerate yeasts were
observed. According to Hankin et al. (1975), the presence of yeasts is a typical indicator of the product lacking freshness. Yeast populations of 106-lo7 cells g-
were observed in cottage cheese by Fleet (1990) during
refrigerated storage of the product, leading to flavour,
odour effects, and gassiness. Recent reviews on the
microbiology of butter (Murphy, 1981) made little
mention of yeasts as potential spoilage organisms,
though lipolytic yeast species, especially species of the
genus Rhodotorula, have been reported to grow on the
surface of butter (Walker and Ayres, 1970; Thomas,
1971) on an irregular basis. Fleet and Mian (1987) were
not able to detect any yeasts in nine of 16 butter samples
examined. In this study we obtained counts of 102-lo3

Comparison of ten media for the enumeration of yeasts

209

Table 1. Yeast populations in dairy products using ten different enumeration media

Mean yeast counts (log)/g or ml

Media

Products
Yoghurt

Raw milk

Butter

Cheddar cheese

Cottage cheese

YMG
Sh

3.310
0.799

3.603
0.085

3.133
0.883

2.680
0.310

2.456
0.55

YML
Sb

3.536
0.761

3.535
0.091

3.450
0.438

2.560
0.364

2.380
0.425

YMGC
Sb

3.176
0.524

4.783
0.905

3.375
0.530

2.640
0.492

2.316
0.411

YMLC
Sb

3.214
0.821

4.7036
0.895

2.856
0.903

2.320
0.476

2.294
0.245

YMGGE
Sb

3.108
0.490

3.816
1.012

2.457
0.915

2.600
0.459

2.000
0.000

OGY
Sb

3.444
0.323

5.226
0.441

2.995
0.708

2.985
1.280

2.388
0.439

YGC
Sb

3.224
0.584

4.673
0.984

3.003
0.967

2.957
0.320

2.161
0.253

RBCA
Sb

3.128
0.775

4.816
0.821

2.896
0.926

2.757
0.258

2.391
0.329

PDA
Sb

3.333
0.632

3.563
0.113

3.295
0.742

2.515
0.350

2.156
0.242

RBC
Sb

3.212
0.492

3.815
0.445

2.880
1.244

2.200
0.282

2.011
0.000

Mean values of triplicate samples (10 samples of each brand).

%tandard error of the mean within each sample of the same product using 10 different samples of the same brand.
The mean value of 10 samples of the same brand (in triplicate).
Means sharing the same letter in the column for raw milk are not significantly different (p 2 0.05).
YMG = Yeast malt extract agar + glucose (pH 3.5); YML = Yeast malt extract agar + lactose (pH 3.5); YMGC = Yeast malt
extract agar + glucose + chloramphenicol (pH 3.5); YMLC=Yeast malt extract agar + lactose + chloramphenicol (pH 3.5);

YMGCE = Yeast malt extract agar + glucose + chloramphenicol + ethanol (pH 3.5); OGY = Oxytetracycline glucose yeast agar;
YGC = Yeast extract glucose chloramphenicol agar; RBCA = Rose bengal chloramphenicol agar; PDA = Potato dextrose agar (pH
3.5); RBC = Rose bengal chlortetracycline

agar.

on the different selective enumeration media.

No significant differences (p > 0.05) among the media
ability to enumerate the yeasts were observed.
The growth of yeasts in Cheddar cheese is expected
due to the ability of various yeasts to grow at a low pH,
low moisture content, elevated salt concentration and
refrigerated storage temperatures (Fleet, 1990; Viljoen
and Greyling, 1995). Yeast counts of lo*-lo3 cells g-
were detected on Cheddar cheese samples. Yeast counts
from the ten samples of Cheddar cheese were not significant (p > 0.05) among the ten selective media in
their ability to enumerate yeasts.
Yoghurt is traditionally prepared by allowing milk to
sour at 4&45C. In recent years, there has been an
enormous increase in the popularity of yoghurt as a
food product, attributed to the use of sugar, fruits and
flavours in its manufacture (Davis, 1970; Kroger, 1976)
which consequently also enhanced yeast spoilage. The
spoilage of yoghurts by yeasts is generally recognized by
the development of yeasty off-flavours, loss of texture
quality due to gas production, and the swelling and
cells g-t

eventual blowing of the product container (Davis, 1970,

1974; Kroger, 1976). Yoghurt should contain no more
than one yeast cell per gram and, if correctly stored
under refrigeration temperatures (5C), a product shelf
life of 3-4 weeks may be expected (Davis, 1970). Table 1
shows the mean log count g-i found in yoghurt.
According to the statistical results based on the yoghurt
samples, no significant differences (p > 0.05) were
obtained among the 10 selective enumeration media.
Because of their low pH, yoghurts create a selective
environment for the growth of yeasts, and the literature
contains several references to the spoilage of yeasts
(Ingram, 1958; Walker and Ayres, 1970; Davis, 1975;
Peppler, 1976; Suriyarachchi and Fleet, 198 1). During
this study, yeast populations of lo3 cells g-i were
obtained which is in agreement with results obtained by
Fleet and Main (1987) who found yeast populations of
lo3 cells g- or more in retail samples of either plain or
fruit yoghurts. However, yeast counts as high as lo6
cells g-i were reported in yoghurts produced in Nigeria
(Green and Ibe, 1987).

210

J. J. Welthagen, B. C. Virjoen

Although milk is the raw material of most dairy products, surprisingly few studies have been conducted on
the specific occurrence of yeasts in either raw or pasteurized milks. Mean counts of 104-lo5 cells ml- were
found in this study on the 10 selective plating media.
Populations less than lo3 cells ml- are reported frequently (Fleet and Mian, 1987), although, yeast counts
as high as lo4 cells ml-i were also reported (Fleet, 1990).
According to the literature, yeast strains rarely grow in
milk during refrigerated storage and are quickly overgrown by psychrotrophic bacteria (Cousin, 1982; Bishop
and White, 1986). However, yeast growth might occur in
milk when bacterial growth is inhibited by residual antibiotics (Fleet and Mian, 1987). Significant differences
(p 5 0.05) among the selective media ability to enumerate yeasts in milk were obtained. Oxytetracycline glucose
yeast agar (OGY) differed from the other media contributing mean counts up to lo5 cells ml-. Yeast extract
glucose chloramphenicol agar (YGC) and rose bengal
chloramphenicol agar (RBCA), both antibiotic-supplemented media also accounted for high yeast counts of
104-lo5 cells ml-. The lowest yeast counts were
obtained with the acidified media, yeast malt extract
agar + glucose (pH 3S)(YM + G), yeast malt extract
agar + lactose (pH 3.5)(YM + G) and potato dextrose
agar (PDA), ranging from 102-lo3 cells ml-. This phenomenon may be indicative that at higher pH values
(milk pH = 6.5), media supplemented with antibiotics
were more suitable for recovering yeasts.
Bacterial growth is restricted by residual antibiotics,
high sugar concentration and low water activity of dairy
products, which contribute to the higher yeast counts
present (Walker and Ayres, 1970). Beuchat and Nail
(1985) revealed that the presence of oxytetracycline
played a more effective role by inhibiting the bacterial
growth compared to the addition of chloramphenicol
and chlortetracycline.
Based on the results obtained in this study, all ten of
the selective media proved to inhibit bacteria and mould
growth to some extent. However, it is generally recognized that antibiotic-supplemented
fungal enumeration
media are superior to acidified media for the enumeration of fungi in foods (Koburger, 1971; Nelson, 1972;
Koburger and Farhat, 1975; Beuchat, 1979). The
increased sensitivity of fungi to acidified media is due, at
least in part, to the presence of injured fungi within the
sample population (Skidmore and Koburger, 1966;
Koburger, 1970; Nelson, 1972). Therefore, as the level
of uninjured fungi in a food sample decreases, the difference in enumeration efficiency between acidified and
antibiotic-supplemented media should decrease (Henson
et al., 1982). Since dairy products are either highly processed, thus virtually free of yeasts (such as dry milk
products), or are processed by means which do not
typically yield sublethally injured fungi, differences
between the use of acidified versus antibiotic media
should be minimal (Beuchat, 1979). This is not the case

as shown by Koburger (1971). In evaluating five dairy

products having a wide range of contamination levels,
Koburger (1971) determined that an acidified medium
yielded only 65% of the counts obtained on antibiotic
amended agar. Results obtained during the present
study showed that significant differences in the enumeration of yeasts associated with dairy products, only
exist at higher pH levels. No significant differences were
found when yeast enumeration was performed on dairy
products of low pH values. However, one must be cautious in making broad recommendations regarding the
use of one medium for all types of dairy products. The
type of yeasts present and the immediate environmental
conditions in the different dairy products differ substantially, thus influencing the suitability of a specific
medium for detecting total yeast populations.

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(Received

31 October

1996; accepted

25 July 1997)