International Journal of Food Microbiology 149 (2011) 194–208

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Review

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus,

Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and
Bifidobacterium spp. in yoghurt — A review
Rabia Ashraf, Nagendra P. Shah ⁎
School of Biomedical and Health Sciences, Faculty of Health Engineering and Science, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, Victoria 8001, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet
Received 7 March 2011 with a recommended level of ≥ 10 6 viable cells/g of a product, assessment of viability of probiotic bacteria in
Received in revised form 23 June 2011 market preparations is crucial. This requires a working method for selective enumeration of these probiotic
Accepted 8 July 2011
bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp.
Available online 21 July 2011
bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could
Keywords:
be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing
Probiotic bacteria fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or
Lactobacillus delbrueckii subsp. bulgaricus reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus
Streptococcus thermophilus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for
Lactobacillus acidophilus selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be
Lactobacillus casei made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Glu) or MRS
Bifidobacterium containing maltose (MRSM) and incubation in a 20% CO2 atmosphere. Lb. casei could be selectively
enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria
(S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei.
Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin,
neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.
© 2011 Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
1.1. Beneficial applications of yoghurt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
2. Differential enumeration of yoghurt cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
2.1. TPPY-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
2.2. RCPB agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
2.3. MRS-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.3.1. MRSB agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.3.2. MMRS-BPB agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.4. Lee's agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.5. LB agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.6. YLA medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
2.7. HHD medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
2.8. BGW agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
3. Selective enumeration of yoghurt cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
3.1. Media for enumerating Lb. delbrueckii subsp. bulgaricus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.1.1. MRS-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.1.2. RCA- based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
3.2. Media for enumerating S. thermophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

⁎ Corresponding author. Tel.: + 61 3 9919 8289; fax: + 61 3 99198284.

E-mail address: Nagendra.Shah@vu.edu.au (N.P. Shah).

0168-1605/$ – see front matter © 2011 Published by Elsevier B.V.

doi:10.1016/j.ijfoodmicro.2011.07.008
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208 195

3.2.1. M17-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

3.2.2. ST agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3. Media for enumerating Lb. acidophilus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3.1. MRS-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3.2. X-Glu agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3.3. BA-based medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
3.3.4. BCP agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.3.5. NA-salicin medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.4. Media for enumerating Lb. casei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.4.1. LC agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.4.2. MRS-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.5. Media for enumerating Bifidobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.5.1. NPNL composite agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.5.2. MRS-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
3.5.3. BLOG agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
3.5.4. CAB medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
3.5.5. RCA-based medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
3.5.6. LP composite agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
3.5.7. TPY-based agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
3.5.8. WCM agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
4. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

1. Introduction monitor survival of probiotic organisms in fermented products has

The health benefits derived by the consumption of foods contain-
ing acidophilus and bifidus products are well documented. The
number of probiotic products is increasing worldwide and it is
estimated that more than 500 probiotic products have been
introduced in the global market over the past decade. Increased
evidence supporting the health-benefits of using probiotic strains has
led to promote commercial interest in developing novel probiotic food
products. Probiotics are non-pathogenic microorganisms mostly of
human origin which, when administered in adequate amounts, confer
a health benefit on the host and are able to prevent or improve some
diseases (Fric, 2007). Following recommendations of a FAO/WHO
working group on the evaluation of probiotics in food (2002), the
suggested definition of probiotics is ‘live microorganisms which when
administered in adequate amounts confer a health benefit on the
host’.
Yoghurt is one of the popular fermented milk product known for
thousands of years and has taken much attention in few years with
the advent of probiotic in dairy products. Lactic acid produced by the
fermentation of lactose acts on milk protein to give yoghurt its
characteristics texture and taste. Being nutritionally rich in protein,
calcium, riboflavin, vitamin B6 and vitamin B12, yoghurt is considered
to have more nutritional benefits than milk. It has a long history of
medicinal uses particularly, for a variety of gastrointestinal diseases
and diarrhoea. In recent years, yoghurt has been used as a vehicle for
probiotic bacteria. In order to obtain health benefits from probiotic
products, recent trend is to use 5 to 6 different strains of probiotic
organisms added with two starter bacterial cultures of S. thermophilus
and Lb. delbrueckii subsp. bulgaricus. B. bifidum, B. breve, B. longum and
B. animalis are commonly used for the production of fermented milks,
in combination with other lactic acid bacteria. B. animalis subsp. lactis
Bb12 has been found to remarkably survive passage through the
gastrointestinal tract and is capable of adhering extraordinarily to
enterocytes (Haschke et al., 1998). Moreover B. animalis subsp. lactis
Bb12 is also a technologically suitable strain to be used as an adjunct
culture in probiotic products since it does not have any adverse effects
on flavour, appearance or taste of the foods in which it is used and
remain viable in high concentrations until the probiotic product is
consumed (Möller and De Vrese, 2004).
Current recommendation level for probiotic bacteria is ≥10 6 cfu/g
of a product. However, studies have shown low viability of probiotics
in market preparations (Shah, 2000; Shah et al., 1995). The need to
often been neglected, with the result that a number of products reach
the market containing low concentration of viable bacteria (Shah
et al., 1995). In order to assess product quality, an important
parameter in monitoring viability and survival of probiotic bacteria
is the ability to count these bacteria differentially. This necessitates a
routine method that could determine the initial inoculum and
estimate the storage time period these organisms remain stable and
consequently a working method for selective enumeration of these
probiotic organisms. Several media for selective enumeration of
probiotic bacteria have previously been proposed and some of these
methods have been reviewed in past (Roy, 2001; Shah, 2000).
However, most of these methods are based on pure cultures of these
organisms and fails to work whilst working with products because the
presence of multiple and closely related species in these products
makes the differential or selective enumeration of probiotic and
starter bacteria difficult due to similarity in growth requirements and
overlapping biochemical characteristics of the species. The develop-
ment of new selective media has been impeded by the recent use of
culture-independent methods and as well as polyphasic approach
(Lahtinen et al., 2006; Masco et al., 2007; Tsai et al., 2008).
1.1. Beneficial applications of yoghurt
Yoghurt has long history of recognition as a dietary product with
many desirable effects. It is made from the symbiotic growth of
S. thermophilus and Lb. delbrueckii subsp. bulgaricus. These yoghurt
starter bacteria do not survive the gastric passage or colonise the gut
and consequently do not play a role in the human gut. Hence, the
recent trend is to add Lb. acidophilus, Bifidobacterium spp. and Lb. casei
to yoghurt. Modzelewska-Kapituła et al. (2008) investigated the
possibility of using two probiotic strains of Lb. plantarum and Lb.
fermentum in yoghurt production. These strains did not acidify milk
during 24 h and 72 h fermentation at 37 °C, but grew well and
remained at the level of 10 8 cfu/mL during 21 days of cold storage. The
authors proposed their application to yoghurt production along with
commercial starter culture viz., Lb. delbrueckii subsp. bulgaricus and S.
thermophilus in obtaining products with typical sensory properties,
pH values and numbers of potentially probiotic bacteria at desired
level of 10 7 cfu/mL.
With the growing popularity of yoghurt as probiotic carrier food,
yoghurt is emerging as a new therapeutic leading edge with different
diseases. A number of health benefits have been claimed for probiotic
196 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
bacteria such as Lb. acidophilus, Bifidobacterium spp. and Lb. casei.
These include antimutagenic properties (LeBlanc et al., 2002; Leu
et al., 2005), anticarcinogenic properties (Wollowski et al., 2001),
anti-diarrhoeal properties (Clancy, 2003; Rowland, 1999; Van Niel et
al., 2002), immune system stimulation (Galdeano and Perdigón, 2006;
Hai et al., 2009; Hsieh and Chou, 2006; Paineau et al., 2008; Prescott
et al., 2008), prevention of eczema and atopy (Isolauri, 2004; Isolauri
et al., 2000; Wickens et al., 2008), reduction in blood pressure,
reduction in serum cholesterol concentration (Hlivak et al., 2005),
increased resistance to infectious diseases (Rautava, 2007) (Li et al.,
2008), growth stimulation (Ohashi, 2006; Ohashi and Ushida, 2009),
improvement in gastroenteritis/inflammatory bowel disease and
suppression of Helicobacter pylori infection (Jones and Foxx-Orenstein,
2007; Myllyluoma et al., 2005; Ryan et al., 2009; Vandenplas et al.,
2007), maintenance of balanced flora and improvement in lactose
metabolism (De Vrese et al., 2001; Gill, 2003; Rowland, 1999; Shah,
2007).
Dietary supplementation with yoghurt is considered as a simple,
effective, and safe treatment that decreases the incidence and
duration of antibiotic associated diarrhoea (Beniwal et al., 2003).
Yoghurt consumption is also useful to alleviate inflammatory bowel
disease (IBD), which involves two major disorders such as ulcerative
colitis and Crohn's disease. The way by which yoghurt exerts such
effect is suggested to be through the immune mechanisms, with an
increased in IgA + cells, capable of modulating the inflammatory
response with a diminution in CD4+ and CD8+ population and an
increase in the apoptosis of the infiltrative immune cells (Gobbato
et al., 2008). The meta-analysis study performed by Rahimi et al.
(2008) failed to demonstrate the efficacy of probiotics in maintaining
remission and preventing clinical and endoscopic recurrence in
Crohn's disease. The authors suggested using probiotic preparations
containing a mixture Lactobacillus with E. coli or Saccharomyces.
Preliminary human studies and in vitro experiments show the
possible beneficial effect of yoghurt in increasing the eradication rate
of H. pylori infection. In addition, there is ever-increasing evidence of
the beneficial effect of yoghurt containing live and active cultures on
the digestion of lactose in persons with lactose intolerance (Adolfsson
et al., 2004). Conventional yoghurt may exert anti-tumour activity by
a decrease in the inflammatory immune response (Perdigón et al.,
2002) and that the dietary supplementation of yoghurt may also play
a role in modulating cell proliferation, inducing apoptosis during the
development of colorectal carcinoma (Rachid et al., 2002). In normal
hosts yoghurt was able to stimulate the immune cells associated with
the gut (Perdigón et al., 1994). The concentration, bacterial repertoire,
and viability be clearly published on yoghurt labels and physicians
should take caution whilst considering yoghurts as a therapeutic
alternate in acute gastroenteritis in children (Dunlap et al., 2009).
2. Differential enumeration of yoghurt cultures
Enumeration of single strain using a selective medium is easily
carried out. However, simultaneous presence of several species
(starter cultures and probiotic bacteria) in a product matrix, sharing
similar cultural characteristics, can make it difficult to achieve a
differential or a selective colony count of each individual species.
Several media have been developed for differential enumeration
of yoghurt culture organisms (Lb. delbrueckii subsp. bulgaricus and S.
thermophilus) including Lee's agar (Lee et al., 1974), and reinforced
clostridial agar (RCA) adjusted to pH 5.5 (Johns et al., 1978). Similarly,
probiotic cultures can be distinguished from non-probiotic on MRS
agar with glucose replaced by maltose incubated at 37 °C or 42 °C
under anaerobic conditions (Lima et al., 2009), rods can be
differentiated from cocci in yoghurt using yoghurt lactic agar (YLA)
(Matalon and Sandine, 1986) and homofermentative LABs can be
differentiated from heterofermentative LAB strains using homofer-
mentative–heterofermentative differential (HHD) medium (McDonald
et al., 1987). Lactic acid bacteria may also be enumerated and
differentiated from the bifidobacteria using media specific for lactic
acid bacteria, such as tryptone, glucose and beef extract (TGE) agar
(Gomes et al., 1995; Gomes et al., 1998), or incubated modified aerobic
condition (5% CO2) to prevent the growth of the bifidobacteria (El-Zayat
and Osman, 2001; Vinderola and Reinheimer, 2000).
Reddy et al. (1972) developed an agar medium for differential
enumeration of lactic streptococci in pure and mixed cultures. The
medium contained arginine and calcium citrate as substrates and
diffusible (K2HPO4) and undiffusible (CaCO3) buffer systems, non-fat
milk (11% solids) as the sole source of carbohydrate (lactose) and
bromocresol purple as the pH indicator. After 36 to 40 h at 32 °C in a
candle oats jars (could be substituted with CO2 incubator), Lactococcus
lactis subsp. cremoris produced yellow colonies with yellow zones
(indicative of acid production alone), whereas Lc. lactis subsp. lactis
and Lc. lactis subsp. lactis biovar. diacetylactis exhibited white colonies
(indicative of neutralisation of acid by the liberated ammonia). The
acid-induced yellow colour of the indicator in and around Lc. lactis
subsp. cremoris colonies tended to diffuse out rapidly beyond 40 h of
incubation and passing this stage, clarity of differentiation was lost.
On further incubation, Lc. lactis subsp. lactis biovar. diacetylactis
utilised suspended calcium citrate and after 6 days, the citrate-
degrading colonies exhibited clear zone against turbid background,
making them easily distinguishable from colonies of the other two
species. Because the medium is not selective, the authors concluded
that the medium must only be used in pure culture studies medium;
however, it proved suitable for quantitative differential enumeration
of lactic streptococci (Table 1).
2.1. TPPY-based agar
Ghoddusi and Robinson (1996) proposed tryptose proteose
peptone yeast extract (TPPY) agar added with prussian blue, as a
differential agar that allows Lb. delbrueckii subsp. bulgaricus, S.
thermophilus, bifidobacteria and Lb. acidophilus to be enumerated on
one medium. Bifidobacterium gave white colonies, S. thermophilus
formed pale blue colonies surrounded by a thin pale blue zone, Lb.
delbrueckii subsp. bulgaricus produced small white colonies sur-
rounded by wide royal blue zones and Lb. acidophilus produced large
pale blue colonies surrounded by a wide royal blue zone.
TPPY-eriochrome agar compose of tryptose, proteose peptone,
yeast extract, glucose, lactose, tween 80, agar and eriochrome black
T which was developed by Bracquart (1981) for the differential
enumeration of Lb. delbrueckii subsp. bulgaricus and S. thermophilus in
yoghurt. After 24 h incubation, S. thermophilus gave circular or semi-
circular colonies of 1–3 mm diameter with characteristic white-
violet (often with a darker centre) colour and the colonies of Lb.
bulgaricus were transparent, diffuse (4–6 mm diameter) of undefined
shape with irregular edges which became granular with 1 cm in
diameter after 48 h. Hence with a mixed culture of Lb. bulgaricus and S.
thermophilus the colonies were easily differentiated and the growth of
two bacteria was rapid and maximal after 24 h incubation, TPPY-
eriochrome medium was proposed as good as M17 and better
than MRS medium where bacteria did not generally develop before
48 h. It was suggested that rapid enumeration of Lb. delbrueckii
subsp. bulgaricus and S. thermophilus in starters could be done because
of their fast growth on TPPY-eriochrome agar medium. But the
medium was proposed not to be used when a great imbalance exists
between the two cultures because the medium is not selective.
2.2. RCPB agar
Reinforced clostridial prussian blue (RCPB) agar was recom-
mended by Ghoddusi and Robinson (1996) for the differential
enumeration of Lb. delbrueckii subsp. bulgaricus, Streptococcus thermo-
philus and bifidobacteria. Lb. delbrueckii subsp. bulgaricus and S.
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208 197

Table 1
Media used for differential enumeration of Lb. delbrueckii subsp. bulgaricus, S. thermophilus, Lb. acidophilus, Lb. casei and Bifidobacterium in yoghurt.

Medium Basea Differential agent References

TPPY-prussian blue TPPY Prussian blue Ghoddusi and Robinson (1996)

TPPY-erichrome TPPY Erichrome agar Bracquart (1981)
RCPB RCA Prussian blue Ghoddusi and Robinson (1996)
RCPB 5.0 RCA pH 5.0 and prussian blue Rybka and Kailasapathy (1996)
MRSB MRS Bile (0.15% w/v) Vinderola and Reinheimer (2000)
MMRS-BPB MRS Bromophenol blue (0.002%) Lee and Lee (2008)
Lee's agar n/a Bromocresol purple Lee et al. (1974)
LB agar Lactic agar n/a Eliker et al. (1956)
YLA Lactic agar Skim milk (7%) Matalon and Sandine (1986)
HHD TPPY Bromocresol green Camaschella et al. (1998)
BGW Whey Bromocresol green Yamani and Ibrahim (1996)
a
TPPY: Tryptose proteose peptone yeast extract, RCA: Reinforced clostridial agar, MRS: deMan Rogosa Sharpe.

Table 2
Media used for selective enumeration of Lb. delbrueckii subsp. bulgaricus, S. thermophilus, Lb. acidophilus, Lb. casei and Bifidobacterium in yoghurt.

Bacteria Medium Basea Selectivity mediator/supplement References

Lb. delbrueckii subsp. bulgaricus MRSF MRS Fructose, incubation at 45 °C for 72 h. Tabasco et al. (2007)
MRS 5.2 MRS pH 5.2, anaerobic incubation at 45 °C for 72 h. Dave and Shah (1996)
RCA 5.3 RCA pH 5.3, anaerobic incubation at 45 °C for 72 h. Dave and Shah (1996)
RCPB 5.0 RCA pH 5 Rybka and Kailasapathy (1996)
S. thermophilus M17 M17 Lactose, aerobic incubation at 37 °C for 24 h Ravula and Shah (1998)
M17L M17 Lactose, aerobic incubation at 45 °C for 24 h Tabasco et al. (2007)
ST ST pH, aerobic incubation at 37 °C for 24 h Dave and Shah (1996)
Lb. acidophilus MRSM MRS Maltoseb Lankaputhra and Shah (1996)
MRSB MRS Bile (0.15%) Lima et al. (2009)
LC MRS Aerobic or anaerobic incubation at 42 °C. Lima et al. (20090
MRS-salicin MRS Salicin Dave and Shah (1996)
MRS-sorbitol MRS Sorbitol Dave and Shah (1996)
MRS-clindamycin MRS Clindamycin, anaerobic incubation at 37 °C for 72 h Van de Casteele et al. (2006)
X-Glu Rogosa agar 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside Kneifel and Pacher (1993)
BA-sorbitol BA Sorbitol Tharmaraj and Shah (2003)
BAM BA Maltose, anaerobic incubation at 43 °C Tharmaraj and Shah (2003)
BCP n/a Penicillin, sodium pyruvate, salicin Bielecka et al. (2000)
NA-salicin NA Anaerobic incubation at 37 °C for 72 h Lankaputhra and Shah (1996)
Lb. casei LC MRS Ribose (1% w/v) and anaerobic incubation at 27ºCc Ravula and Shah (1998)
MRS-LP MRS LP mixtured, anaerobic incubation at 37 °C for 72 h Vinderola and Reinheimer (1999)
MRSB MRS Bile (0.15% w/v) Vinderola and Reinheimer (1999)
MRS-salicin MRS Salicin Dave and Shah (1996)
Bifidobacterium spp. NPNL BL NPNL solutiond, anaerobic incubation at 37 °C for 72 h Teraguchi et al. (1978)
MRS-NPNL MRS NPNL solutiond, anaerobic incubation at 37 °C for 72 h Dave and Shah (1996)
TOS-NPNL N/A NPNL solutiond Wijsman et al. (1989)
RMS-PPNL RA Sodium propionate, neomycin, paromomycin, LiCl Samona and Robinson (1991)
MRS MRS n/a Dave and Shah (1996)
BSM MRS Cysteine hydrochloride and mupirocin Simpson et al. (2004)
MRS-raffinose MRS Raffinose, LiCl (0.05%) and incubation at 45 °C Tabasco et al. (2007)
MRSD MRS Dicloxacillin Sozzi et al. (1990)
MRSM MRS Maltose Rybka and Kailasapathy (1996)
BLOG BL Oxgall, gentamycin Lim et al. (1995)
MCAP CAB Propionic acid, 5 mL Beerens (1990, 1991)
CAB-NL CAB Neomycin, LiCl Chapon and Kiss (1991)
RAF 5.1 CAB LP mixtured Roy et al. (1997)
MCAB-LG CAB Lactose and gentamycin Roy et al. (1997)
DP CAB Dicloxacillin, propionic acid. Bonaparte et al. (2001)
RCA RCA n/a Dave and Shah (1996)
BIM25 RCA Nalidixic acid, Polymycin B, Iodoacetate, Munoa and Pares (1988)
2,3,5-triphenyltetrazolim chloride
LP LCL LP mixtured Lapirre et al. (1992)
AMC RCA Nalidixic acid, Polymycin B, Iodoacetate, Arroyo et al. (1995)
2,3,5-triphenyltetrazolim chloride, LP mixtured
GL n/a LiCl Iwana et al. (1993)
RB LCL LiCl, sodium propionate Hartemink et al. (1996)
BFM n/a LiCl, Methylene blue, Propionic acid, 5 mL Nebra and Blanch (1999)
MTPY TPY Mupirocin Vlková et al. (2004)
TPYD TPY Dicloxacillin Sozzi et al. (1990)
WCM n/a Mupirocin Rada and Koc (2000)
a
MRS: deMan Rogosa Sharpe, RCA: Reinforced clostridial agar, ST: Streptococcus thermophilus agar, BA: Basal agar, NA: Nutrient agar, BL: Blood-glucose-liver, RA: Rogosa agar,
CAB: Columbia agar base, LCL: Liver-cystine-lactose, and TPY: Trypticase phytone yeast.
b
When Bifidobacterium is not present, however if it is present then MRSM agar could be used to get total counts of Lb. acidophilus and Bifidobacterium.
c
If Lb. rhamnosus was present, total counts of Lb. casei and Lb. rhamnosus could be obtained using MRSV agar at 37 °C and anaerobic incubation for 72 h. To obtain the counts of Lb.
casei, the count of Lb. rhamnosus on MRSV agar incubation at 43 °C for 72 h under anaerobic condition could be subtracted from the total counts of Lb. casei and Lb. rhamnosus.
d
Made from stock solution as follows: LP mixture-LiCl, 2 g/L; sodium propionate, 3 g/L. NPNL solution-neomycin sulphate, 100 mg/L; paromomycin, 200 mg/L; nalidixic acid,
15 mg/L; and LiCl, 3 g/L.
198 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
thermophilus formed pale blue colonies surrounded by wide royal blue
or thin light blue zones whilst B. bifidum and B. adolescentis appeared
as white colonies, respectively. RCPB was also reported to allow the
differential enumeration of S. thermophilus and Lb. delbrueckii subsp.
bulgaricus and bifidobacteria without the presence of other probiotics,
based only on colony characteristics, particularly colour (Onggo and
Fleet, 1993).
Rybka and Kailasapathy (1996) reported the importance of
another version of RCPB for its selectivity for bifidobacteria and Lb.
bulgaricus only and inhibition of non-lactic flora growth due to a low
pH (pH 5.0). They found that B. infantis, B. breve, and B. bifidum
produced white colonies whilst B. longum produced blue ones.
Similarly, on RCPB (pH 5.0), Lb. delbrueckii subsp. bulgaricus develops
white colonies with a wide dark blue halo in contrast to the white
colonies of bifidobacteria. On both media such as RCPB and RCPB (pH
5.0) either colonies did not appear or if they were produced, it was
difficult to distinguish their colonial characteristics on the basis of
visual examination. In addition these media have not been validated
with Lb. casei which could have added errors in the accurate
enumeration of Bifidobacterium spp.
Grosso and Fávaro-Trindade (2004) evaluated RCPB for enumer-
ating B. lactis in yoghurt. S. thermophilus, and Lb. delbrueckii subsp.
bulgaricus and B. lactis were easily differentiated in RCPB by the
distinct morphologies of their colonies. Lb. delbrueckii subsp. bulgar-
icus grew forming colonies with diameters of 2–3 mm, each with a
small white clearly defined centre surrounded by a relatively large
blue halo. S. thermophilus grew forming colonies with white centres
(less clearly defined than those of Lb. delbrueckii subsp. bulgaricus)
and a blue halo with a diameter of about 1 mm. B. lactis formed very
small cylindrical white colonies (approximately 0.5 mm in diameter).
The results were similar to that reported by Onggo and Fleet (1993),
with the difference that these authors obtained larger colonies for S.
thermophilus than for Lb. delbrueckii subsp. bulgaricus. This difference
was attributed to the use of strains from different companies, so from
different origins. After 7 days of storage counting of B. lactis in RCPB
became impossible, because the colonies of S. thermophilus and Lb.
delbrueckii subsp. bulgaricus dominated the whole plate, since their
numbers were much higher than that of B. lactis.
2.3. MRS-based agar
2.3.1. MRSB agar
Mortazavian et al. (2007) evaluated the suitability of MRS-bile
(MRSB) agar for enumeration of mixed probiotic cultures in cultured
dairy products and reported that MRSB agar showed a good suitability
for differential enumeration of probiotic bacteria in the presence of
Lb. acidophilus, Lb. casei and yoghurt bacteria under both aerobic and
anaerobic conditions. Differential and selective enumerations of
probiotics was suitable on MRSB from mixed culture composition
containing bifidobacteria, Lb. casei and yoghurt bacteria under anaero-
biosis and aerobiosis, respectively. Selective enumeration of only
Lb. acidophilus could be made on MRSB agar from mixed culture
containing bifidobacteria and yoghurt bacteria under aerobiosis.
Furthermore, by using the subtractive enumeration method based on
the subtraction of the colonies growth under aerobic condition (only Lb.
acidophilus) from the colonies growth under anaerobic condition (both
Lb. acidophilus and bifidobacteria), the number of bifidobacteria in the
presence of Lb. acidophilus and yoghurt bacteria could also be well
determined. Dave and Shah (1996) found that recovery of Lb. acidophilus
was one or two log cycles lower on MRSB agar compared with other media
tested. Similarly, recovery of Bifidobacterium was also low and varied on
MRSB, so the medium was not recommended for enumeration purpose.
2.3.2. MMRS-BPB agar
Modified MRS agar containing bromophenol blue (MMRS-BPB)
was evaluated by Lee and Lee (2008) for isolation and enumeration of
each LAB from fermented foods in mixed culture. Comparison of
MMRS-BPB was made with MMRS and plate count agar with
bromocresol purple (PCA-BCP). MMRS was used for the non-selective
enumeration of lactobacilli as well as bifidobacteria and BPB (0.002%)
was used as pH indicator to detect pH values from 3 to 5 developed by
LAB by producing yellow colour at pH 3.0 and violet at pH 4.6.
Colonies appeared on the medium were much easier to differentiate
compared with those produced on MMRS because of the variation in
sizes and colours. The authors proposed advantages of using MMRS-
BPB for enumeration of LABs as it takes less incubation time than PCA-
BCP, it supports growth of all LABs including bifidobacteria and it
allows differentiation of each LAB in a mixed culture.
2.4. Lee's agar
Lee's agar, described by Lee et al. (1974) is used for the differential
enumeration of yoghurt starter bacteria and the medium is also
recommended by APHA for the same purpose (Downes and Ito, 2001).
Matalon and Sandine (1986) found that differential rod–coccus
enumeration on Lee's agar based on colony colour is difficult. On
Lee's agar, S. thermophilus formed yellow, smooth, discrete colonies
with entire edges, whereas Lb. delbrueckii subsp. bulgaricus gave rise
to pale yellow, flat, larger colonies with irregular or serrated edges.
Briceño and Martínez (1995) found the counts in the Lee's agar
showed a better recuperability of lactic acid bacteria in yoghurts. In
another study, Lee's agar supported the growth of Lb. acidophilus and
B. longum incubated at 37 °C for 48 h under microaerophilic
conditions (Zacarchenco and Massaguer-Roig, 2004).
Lee's agar contains sucrose, which is fermented by S. thermophilus but
not by most of Lb. delbrueckii subsp. bulgaricus strains, and lactose, which
is utilised by both species. With a suitable combination of sucrose and
lactose, the rate of acid production by S. thermophilus is enhanced and
that of Lb. bulgaricus restricted. Therefore, streptococci grow first and
produce a creamy, buttery aroma from diacetyl and similar metabolites.
The redox potential is also thus, lowered by streptococci, which enables
lactobacilli to grow, thereby growth stimulatory products for streptococci
are synthesised by lactobacilli. Hence the typical sharp acetaldehyde
flavour of mature yoghurt is formed. Casein enzymic hydrolysate and
yeast extract provide the essential nitrogenous nutrients to the yoghurt
starter bacteria. Lactose and sucrose are the fermentable carbohydrates.
Calcium carbonate along with the dipotassium phosphate is added to
buffer the medium and avoid the drastic drop in pH due to lactic acid
formation. Bromocresol purple is the pH indicator, which turns yellow in
acidic condition and imparts yellow colour to the colony.
2.5. LB agar
Lactobacillus (LB) agar also named as Eliker's lactic agar is general
purpose agar specifically designed for the enumeration of streptococci
and lactobacilli as starter cultures in dairy products. It consisted of
tryptone (2.0%), yeast extract (0.5%), gelatin (0.2%), glucose (0.5%),
sucrose (0.5%), lactose (0.5%), sodium chloride (0.4%), sodium acetate
(0.15%) and ascorbic acid (0.05%). The medium allows separation of
S. thermophilus and Lb. delbrueckii subsp. bulgaricus based on colony
morphology. Matalon and Sandine (1986) revealed poor or no growth
of some strains of S. thermophilus strains on this medium. Lactic agar
supplemented with tween 80 (0.1%) for stimulation of the lactobacilli,
supported excellent growth of both S. thermophilus and Lb. delbrueckii
subsp. bulgaricus in mixed and single cultures and commercial
yoghurts but authors found it difficult to differentiate both bacteria
based on colony morphology.
2.6. YLA medium
Similar metabolic characteristics of S. thermophilus and Lb.
delbrueckii subsp. bulgaricus make difficult the development of a
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
reliable rod–coccus differential agar. Matalon and Sandine (1986)
considered acid production to be a reliable trait for this purpose. They
used addition of 7% skim milk (11% solids) to lactic agar in place of
2,3,5-triphenyltetrazolium chloride and it allowed good rod–coccus
differentiation, regardless of strain or yoghurt brand. The medium was
named as yoghurt lactic agar (YLA) and it produced large white
colonies of Lb. delbrueckii subsp. bulgaricus surrounded by a distinctive
cloudy zone and smaller white colonies of S. thermophilus devoid of a
surrounding halo. It was suggested that acid precipitation of casein
was responsible for halo effect, which was more evident in the
colonies of Lb. delbrueckii subsp. bulgaricus because of significantly
higher production of acid than S. thermophilus.
2.7. HHD medium
McDonald et al. (1987) designed homofermentative–heterofer-
mentative (HHD) medium that allowed differential enumeration of
homofermentative and heterofermentative LAB. Species of Strepto-
coccus, Lactobacilli, Leuconostoc and Pediococcus were used in the
study. The medium composed of fructose, KH2PO4, trypticase peptone,
phytone peptone, casamino acids, yeast extract, tween 80, bromocre-
sol green and agar, where bromocresol green was used as pH indicator
and limited amount of fructose as the only source of carbohydrate.
HHD broth inoculated with homofermentative LAB was green, whilst
HHD broth containing heterofermentative LAB remained blue. After
3 days, HHD agar incubated at 30 °C produced blue to green colonies
of homofermentative LAB species whilst heterofermentative colonies
remained white. By using HHD agar, the authors identified nine
heterofermentative species, of these six were of Lactobacillus spp. and
three were of Leuconostoc spp. Twelve homofermentative species of
LAB were also identified comprised of four species of streptococci, one
of pediococci and seven of lactobacilli. Since the study was based on
pure cultures alone and in combinations, so the medium needs to be
validated before using commercial strains and products.
Camaschella et al. (1998) determined the suitability of HHD agar
for the detection and specific enumeration of S. thermophilus, Lb.
delbrueckii subsp. bulgaricus, Lb. acidophilus and bifidobacteria on the
same plate. Colonies of S. thermophlius exhibited on HHD agar were of
two different and distinguishable morphologies; one type of colonies
were small, circular, smooth and transparent and other type of
colonies were circular, convex and dark green coloured. Colonies of
bifidobacteria were similar in size to those of S. thermophilus, but they
appeared translucent, convex like a drop of water. Colonies of Lb.
delbrueckii subsp. bulgaricus were large, of irregular in shape, with flat
surface, regular bright green with internal undulatory streaking.
Whereas colonies of Lb. acidophilus were large, of irregular shape,
convex and pyramid shaped surface, light brown with a small central
spot of dark green colour. The use of HHD medium allowed the 4
different species to be distinguished and counted separately on the
same plate. Counts on HHD were slightly but significantly higher than
those on reference media (MRS, MRS 5.4, MRSD, M17) for the 4
species. The medium and method offer the advantage in terms of
economic feasibility of using only one culture medium to enumerate
S. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and
bifidobacteria in mixed cultures containing these species in varying
amount. This allows saving of material and rapid performance for
routine controls of dairy and probiotic products.
2.8. BGW agar
An improved whey medium namely bromocresol green whey
(BGW) agar was developed for the differential enumeration of Lb.
delbrueckii subsp. bulgaricus and S. thermophilus and the medium was
compared with MRS agar and M17 agar for enumerating these
bacteria in commercial yoghurt and labneh (traditional dairy product
in Jordon) by Yamani and Ibrahim (1996). BGW agar composed of two
199
parts of whey obtained from reconstituted non-fat dry milk (NFDM)
(15% w/w, pH 5.7) and one part of a sterile agar solution containing
yeast extract (3%), K2HPO4 (1.2%), bromocresol green (0.004%) and
agar (4%). Colonies of Lb. delbrueckii subsp bulgaricus in BGW agar
were larger, light in colour with greenish centres (or in some green
colour extended from centres) and in the form of an irregular mass
with twisted filament projections, whilst colonies of S. thermophilus
were green, small, compact, regular, lenticular with entire edges
sometimes with white margins. For 21 pure cultures of each of these
bacteria, BGW agar performed significantly better than MRS agar in
seven (33.3%) cultures of Lb. delbrueckii subsp. bulgaricus and better
than M17 agar in 14 (66.6%) cultures of S. thermophilus. In 20 yoghurt
samples examined, BGW agar performed significantly better than
MRS in five (25.0%) samples, whilst MRS agar performed significantly
better than BGW agar in the remaining four (20.0%) samples. BGW
agar performed significantly better than M17 agar in nine (45.0%)
samples, and M17 performed no better than BGW agar in any of the
samples. Wider ranges in counts were noted in MRS agar and M17
agar than those of BGWA. In the labneh samples analysed immedi-
ately after packaging, BGW agar performed significantly better than
MRS in two (22.2%) samples and better than M17 in six (66.6%)
samples. MRS agar performed significantly better than BWG agar in
the other two (22.2%) samples. A slight decrease in the counts of
yoghurt bacteria of labneh samples was observed after 14 days at 7 °C
that was attributed to high degree of resistance of yoghurt culture
bacteria to acidity and refrigeration.
3. Selective enumeration of yoghurt cultures
Whilst dealing with similar type of bacteria in product like
yoghurt, differential enumeration of probiotic bacteria and lactic
acid bacteria becomes difficult. Therefore, the viability and survival of
these bacteria can be assessed via selective enumeration methods.
Tharmaraj and Shah (2003) evaluated nineteen bacteriological media
to assess their suitability to selectively enumerate Lb. delbrueckii
subsp. bulgaricus, S. thermophilus, Lb. casei, Lb. rhamnosus, Lb.
acidophilus, Bifidobacterium, and Propionibacterium. Of the media
and incubation conditions assessed, S. thermophilus (ST) agar and
aerobic incubation at 37 °C for 24 h suited best for S. thermophilus,
MRS agar (pH 4.58 or pH 5.20) and anaerobic incubation at 45 °C for
72 h were suitable for Lb. delbrueckii subsp. bulgaricus, MRS-
vancomycine agar (MRSV) and anaerobic incubation at 37 °C for
72 h were suitable for Lb. rhamnosus, MRSV agar and anaerobic
incubation at 37 °C for 72 h were selective for Lb. casei, MRS agar at
43 °C for 72 h or basal agar (BA) supplemented with maltose at 43 °C
for 72 h or BA-sorbitol agar at 37 °C for 72 h, under anaerobic
incubation were found best for Lb. acidophilus, MRS-NPNL agar under
anaerobic incubation at 37 °C for 72 h for Bifidobacterium.
Similarly, several selective media have been developed for
enumeration of pure cultures of Bifidobacterium spp. including NPNL
agar (Arroyo et al., 1994; Burford, 1989; Laroia and Martin, 1991;
Onggo and Fleet, 1993; Teraguchi et al., 1978). For selective
enumeration of bifidobacteria in dairy products, existing medium
bases are often supplemented with elective and selective agents such
as Columbia agar base (CAB) media are supplemented with lithium
chloride and sodium propionate plus raffinose, CAB media added with
propionic acid and dicloxacillin or MRS supplemented with NPNL.
However, these media may not be suitable for selective enumeration
of Bifidobacterium spp. in the presence of Lb. acidophilus and yoghurt
culture organisms. Further, differences exist amongst the strains of
the same species with respect to sugar fermentation characteristics
and tolerance to low pH, and bile. There is a growing concern that
some media that contain bile or antibiotics might also restrict the
growth of Lb. acidophilus or Bifidobacterium and that counts obtained
are not representative of the actual number of viable cells present in
the product.
200 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
A spread plate method using four different agars, MRS, acidified
MRS, MRS with triphenyl tetrazolium chloride (TTC) and Bifidobacter-
ium selective medium (BSM), was evaluated for the selective
enumeration of probiotic bifidobacteria in animal feed by Leuschner
et al. (2002). The use of MRS agar supplemented with cysteine
hydrochloride (the selective bifidobacteria medium without antibi-
otics) was recommended for routine analysis of products containing
only bifidobacteria. Depending on the presence and concentration of
other probiotics such as enterococci, lactobacilli and pediococci,
acidified MRS or MRS + TTC agar was recommended. For selective
enumeration of bifidobacteria from products containing less number
of bifidobacteria compared with other microorganisms, BSM was
reported as selective media (Leuschner et al., 2002; Leuschner et al.,
2003) (Table 2).
3.1. Media for enumerating Lb. delbrueckii subsp. bulgaricus
3.1.1. MRS-based agar
3.1.1.1. MRSF agar. Tabasco et al. (2007) discovered that a non-
antibiotic medium based on the acidified MRS medium recommended
by ISO/FDIS 7889 IDF 117 standard on enumeration of yoghurt
characteristic microorganisms (ISO, 2002), was not selective for
enumeration of Lb. delbrueckii subsp. bulgaricus since it also allowed
growth of Lb. acidophilus, Lb. paracasei subsp. paracasei, and B. lactis.
They proposed the use of MRS containing fructose (MRSF) for this
purpose which comprised of MRS fermentation broth, exclusive of
glucose or meat extract and enriched with 0.2% tween 80 and
supplemented with 1% fructose, 0.8% casein acid hydrolysate, 0.05%
cysteine and 1.5% agar. The method was found to be differential
against Lb. acidophilus when the medium was enriched with 0.2%
between 80, showing a clear morphological differentiation between
lenticular colonies with 1–2 mm diameter of Lb. delbrueckii subsp.
bulgaricus and cottony–fluffy colonies with 2–3 mm diameter of Lb.
acidophilus. Incubation of the medium at 45 °C and replacement
of glucose by fructose excluded Lb. paracasei subsp. paracasei and
B. lactis, respectively. MRSF was found to be suitable for enumeration of
Lb. delbrueckii subsp. bulgaricus from mixed cultures in fermented milk
when incubation is carried out at 45 °C for 72 h (Tabasco et al., 2007).
3.1.1.2. MRS 5.2 agar. The MRS agar at pH 5.2 (MRS 5.2) was
recommended for the enumeration of Lb. delbrueckii subsp. bulgaricus
when the incubation is carried out at 45 °C for 72 h, although
occasional growth of Bifidobacterium spp. was recorded (Dave and
Shah, 1996; Lankaputhra and Shah, 1996). Lb. delbrueckii subsp.
bulgaricus could be enumerated using MRS agar (pH 4.58 or pH 5.2)
and under anaerobic incubation at 45 °C for 72 h. When the pH of
MRS agar was reduced to 5.2 and the incubation temperature was
increased to 43 °C, only Lb. delbrueckii subsp. bulgaricus (which
formed 1.0 mm, white rough irregular colonies), Lb. rhamnosus
(which formed 2 mm, shiny smooth white colonies) and Lb.
acidophilus (which formed 0.1 to 0.5 mm, brown, rough irregular
colonies) showed good growth. MRS agar at pH 5.2, under anaerobic
incubation at 43 °C could be selective for Lb. delbrueckii subsp.
bulgaricus if Lb. rhamnosus and Lb. acidophilus were not present in a
product. The colony morphology of Lb. delbrueckii subsp. bulgaricus
and Lb. rhamnosus was very different and these two organisms could
be easily differentiated if Lb. rhamnosus was present in the product.
Therefore, pH modified MRS agar (pH 4.58) and anaerobic incubation
at 43 °C could be used to selectively enumerate Lb. delbrueckii subsp.
bulgaricus from a product (Tharmaraj and Shah, 2003). Similarly, a
series of culture media was evaluated for selective enumeration of 10
commercial probiotic cultures by Van de Casteele et al. (2006) and
MRS 5.2 was concluded as the most optimal media for enumeration of
Lb. delbrueckii subsp. bulgaricus.
3.1.2. RCA- based agar
3.1.2.1. RCA 5.3 agar. Reinforced clostridial agar at pH 5.3 (RCA 5.3)
was recommended for the enumeration of Lb. delbrueckii subsp.
bulgaricus when the incubation is carried out anaerobically at 45 °C for
72 h (Dave and Shah, 1996). Some of the strain of bifidobacteria also
grew on this media but the colonies formed by Lb. delbrueckii subsp.
bulgaricus were easily differentiated from those of bifidobacteria. In a
separate experiment, recovery of Lb. delbrueckii subsp. bulgaricus was
highest on RCA (6.8), followed by recovery on MRS agar. The growth
of bacteria was partially inhibited when pH of RCA and MRS agar was
lowered to 5.8, and then it was further inhibited at pH 5.2. On the basis
of results obtained, RCA 5.3 with incubations carried out anaerobically
at 45 °C for 72 h, was found suitable for selective recovery and
counting of Lb. delbrueckii subsp. bulgaricus (Dave and Shah, 1996).
3.1.2.2. RCPB 5.0 agar. Rybka and Kailasapathy (1996) found
reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) selective
for bifidobacteria and Lb. bulgaricus only because it inhibited growth
of non-lactic flora due to its low pH. The authors discovered that RCPB
5.0 agar selected only Lb. delbrueckii subsp. bulgaricus from ‘classical’
yoghurt. Bifidobacterium medium (BFM), a selective media for
Bifidobacterium proposed by Nebra and Blanch (1999) was reported
to give lower level of recovery of Lb. delbrueckii subsp. bulgaricus as
well. The medium comprised of meat extract (2 g/L), yeast extract
(7 g/L), starch (2 g/L), L-cysteine hydrochloride (0.5 g/L), sodium
chloride (5 g/L), peptone (5 g/L), tryptone (2 g/L), lactulose (5 g/L),
riboflavin (1 mg/L), thiamine chloride hydrochloride (1 mg/L), methy-
lene blue (16 mg/L), lithium chloride (2 g/L), propionic acid (99%; 5 mL/
L) and agar (15 g/L). Propionic acid, lithium chloride, and methylene
blue inhibited the growth of some related bacterial species and the low
pH (5.5) of BFM inhibited the growth of Enterobacteriaceae.
3.2. Media for enumerating S. thermophilus
3.2.1. M17-based agar
3.2.1.1. M17 agar. M17 agar is recommended by the International Dairy
Federation (IDF, 1981) for selective enumeration of S. thermophilus
from yoghurt. M17 agar is also recommended by APHA for the
cultivation of lactic streptococci (Downes and Ito, 2001). M17 agar
found to be selective for S. thermophilus (Ravula and Shah, 1998).
Shankar and Davies (1977) reported the use of M17 for isolation
and enumeration of S. thermophilus from yoghurt. M17 containing β-
glycerophosphate was found inhibitory to many strains of
Lb. delbrueckii subsp. bulgaricus but it supported good growth of
S. thermophilus, so the medium was used for selective enumeration
S. thermophlius from yoghurt. The media can also be used for cultivation
and maintenance of starter cultures for cheese and yoghurt
manufacturing. In addition this medium helps in detecting streptococ-
cal mutants that are lactose non-fermenters. A series of culture media
was evaluated for selective enumeration of 10 commercial probiotic
cultures by Van de Casteele et al. (2006). All yoghurt starters displayed
a good recovery of S. thermophilus on M17 medium so the authors
reported M17 the most suitable media to enumerate the yoghurt starter
viz. S. thermophilus.
3.2.1.2. M17L agar. Tabasco et al. (2007) proposed the selective plating
methods for enumeration and identification of mixed cultures of
S. thermophilus, Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb.
paracasei subsp. paracasei and B. lactis in fermented milk products
based on selective antibiotic-free media and different incubation
conditions. Incubation under aerobic conditions at 45 °C for 24 h in
M17 agar supplemented with lactose (M17L) was found suitable for
selective enumeration of S. thermophilus, since the incubation
conditions prevented the growth of Lb. paracasei subsp. paracasei
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
found at 37 °C and that of Lb. delbrueckii subsp. bulgaricus and B. lactis
that developed under anaerobic conditions.
3.2.2. ST agar
ST agar at 37 °C for 24 h and aerobic condition were selective for
S. thermophilus amongst the media tested by Tharmaraj and Shah
(2003). S. thermophilus formed tiny colonies of around 0.1–0.5 mm
diameter, on ST agar at 37 °C under aerobic incubation after 24 h. This
incubation time was insufficient for other cultures to grow even if
these were not inhibited completely (Dave and Shah, 1996; Tharmaraj
and Shah, 2003).
3.3. Media for enumerating Lb. acidophilus
Several media have been suggested for the enumeration of
Lb. acidophilus, including bile medium (Collins, 1978), Rogosa agar,
MRS containing maltose (MRSM) (Lankaputhra and Shah, 1996),
raffinose or melibiose in place of dextrose (Hull and Roberts, 1984),
cellobiose–esculin agar (Hunger, 1986), and agar medium based on X-
Glu (Kneifel and Pacher, 1993). Some of these and others have been
discussed as follows.
3.3.1. MRS-based agar
3.3.1.1. MRS agar. MRS agar under aerobic or anaerobic incubation at
43 °C for 72 h could be used to count Lb. acidophilus (except DS 910), if
Lb. delbrueckii subsp. bulgaricus and S. thermophilus are not present in
the product (Tharmaraj and Shah, 2003). Lb. acidophilus formed
smaller rough brownish colonies of 0.1 to 1.0 mm diameter which
could be easily differentiated from smooth shiny disc like colonies
(2.5 mm diameter) of Lb. rhamnosus. Since Lb. delbrueckii subsp.
bulgaricus and S. thermophilus also grew on MRS agar, enumeration of
Lb. acidophilus from commercial yoghurts cannot be carried out on
MRS agar (Tharmaraj and Shah, 2003).
3.3.1.2. MRSM agar. Lankaputhra and Shah (1996) developed a simple
method for selective enumeration of Lb. acidophilus in the presence of
yoghurt starter bacteria and Bifidobacterium spp. based on sugar
fermentation patterns. The authors proposed the use of MRS-maltose
(MRSM) agar for selective enumeration of Lb. acidophilus in the
presence of yoghurt organisms in a product, which does not contain
Bifidobacterium spp. They also proposed use of MRSM agar for
enumerating total counts of Lb. acidophilus and Bifidobacterium.
MRSM was also proposed by Tabasco et al. (2007) for differentiation
between Lb. acidophilus and Lb. paracasei. Selective enumeration of Lb.
acidophilus against S. thermophilus and B. lactis in fermented milk
using MRSM and incubation in a 20% CO2 atmosphere were found to
be selective against Lb. delbrueckii subsp. bulgaricus and differential
against Lb. paracasei subsp. paracasei.
In another study by Lima et al. (2009) replacement of glucose in MRS
agar by maltose (MRSM) or trehalose (MRST) affected the growth of the
yoghurt bacteria (RICH), represented the essentiality of glucose for their
growth, regardless of the temperature and atmosphere of incubation.
However, Rybka and Kailasapathy (1996) found differences in colony
size of Lb. acidophilus and B. bifidum which allowed the isolation and
differentiation of these two species on MRSM agar.
3.3.1.3. MRSB agar. Lima et al. (2009) proposed the best media i.e. MRS
agar with bile salts (MRSB) and best incubation conditions i.e. 37 °C or
42 °C, aerobiosis for enumerating Lb. acidophilus. However, Talwalkar
and Kailasapathy (2004) categorised MRSB as unselective in final
identification and enumeration of probiotic bacteria.
3.3.1.4. LC agar. For enumeration of Lb. acidophilus in a mixed culture,
the media and incubation conditions recommended by Lima et al.
(2009) were Lb. casei (LC) medium, 42 °C, aerobiosis or anaerobiosis.
201
Additional plating in MRS agar (or MRS 5.4 agar) will lead to counts of
S. thermophilus and Lb. delbrueckii subsp. bulgaricus, which corre-
sponded to the difference in the counts in the two media.
3.3.1.5. MRS-salicin or MRS-sorbitol agar. MRS-salicin and MRS-
sorbitol agars were developed for selective enumeration of Lb.
acidophilus by Dave and Shah (1996). In their study, MRS-salicin
and MRS-sorbitol agars supported the growth of Lb. acidophilus and
inhibited the growth of bifidobacteria. Similarly, S. thermophilus and
Lb. delbrueckii subsp. bulgaricus did not grow on these media.
However if Bifidobacterium spp. grew on these media, the colonies
were easily distinguishable from those of Lb. acidophilus. The results
mimic to the findings of Hull and Roberts (1984) that replacing
dextrose in MRS agar with maltose and salicin, the recovery of Lb.
acidophilus was almost the same. Dave and Shah (1996) found the use
of salicin uneconomical for the routine testing, and, hence they
proposed the use of sorbitol in MRS-sorbitol agar, economical and
selective for differential enumeration of Lb. acidophilus. In another
study by Ravula and Shah (1998), Lb. casei was reported to grow in
MRS-salicin agar, so they concluded that the medium cannot be used
for selective enumeration of Lb. acidophilus in products containing
both Lb. casei and Lb. acidophilus. MRS-sorbitol agar (Dave and Shah,
1996) was found to be unselective for the enumeration of Lb.
acidophilus for the commercial yoghurts tested in the study by
Talwalkar and Kailasapathy (2004).
3.3.1.6. MRS-clindamycin agar. MRS-clindamycin agar was considered
as the preferred medium for the selective enumeration of commer-
cial Lb. acidophilus strains La-145 and Lafti L10 used in the study by
Van de Casteele et al. (2006). International Organization for
Standardization (ISO, 20128/IDF 192: 2006) (ISO, 2006) has
recommended MRS-clindamycin agar for the enumeration of
presumptive Lb. acidophilus in milk products including fermented
and non-fermented milks, milk powders and infant formula where
presumptive Lb. acidophilus strains are present along with other
lactic acid bacteria and bifidobacteria.
3.3.2. X-Glu agar
Kneifel and Pacher (1993) developed an agar medium, designat-
ed X-Glu agar, for the selective enumeration of Lb. acidophilus in
yoghurt-related milk products containing mixed microflora of
lactobacilli, streptococci and bifidobacteria. The detection principle
was based on the specific visualisation of the β-D-glucosidase
activity of Lb. acidophilus via a chromogenic reaction of 5-bromo-4-
chloro-3-indolyl-β-D-glucopyranoside which is incorporated into a
Rogosa agar medium at 40 μg/mL. The X-Glu agar was found to
more selective than other commonly used media (MRS, TGV,
conventional Rogosa agar) in tests involving different bacterial test
strains, yoghurt and yoghurt-related products (Kneifel and Pacher,
1993).
3.3.3. BA-based medium
3.3.3.1. BA-sorbitol medium. Highest recovery of Lb. acidophilus was
found in basal agar (BA) supplemented with 2% w/v of sorbitol
amongst the media evaluated for selective enumeration of Lb.
acidophilus in the study by Tharmaraj and Shah (2003). All strains of
Lb. acidophilus tested formed rough dull, small, and brownish colonies
which could be easily enumerated and differentiated and from shiny,
large, smooth and white colonies of Lb. casei and Lb. rhamnosus.
3.3.3.2. BAM medium. When Lb. delbrueckii subsp. bulgaricus is present
in the product, basal agar supplemented with maltose (BAM) under
anaerobic incubation at 43 °C could be used and only rough dull,
small, and brownish colonies should be enumerated as Lb. acidophilus
(Tharmaraj and Shah, 2003).
202 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
3.3.4. BCP agar
High recovery (89–100%) of Lb. acidophilus was found in minimal
nutrient agar termed as bromocresol purple (BCP) agar, along with
fine visibility of colonies by surrounding yellow aureole. The medium
was supplemented with penicillin and sodium pyruvate as selective
agent and glucose replaced with 0.5% or 1.0% salicin. BCP agar also
prevented the formation of colonies by concomitant bacteria and
hence it was chosen by Bielecka et al. (2000) as the most suitable
media for selective enumeration of Lb. acidophilus in bio-yoghurts.
3.3.5. NA-salicin medium
Nutrient agar supplemented with salicin (NA-salicin) did not
support growth of S. thermophilus or Lb. delbrueckii subsp. bulgaricus
in the report of Lankaputhra and Shah (1996) and this finding was
confirmed in another study by Van de Casteele et al. (2006) for the
starter (Delvo Yog CY221) and reference strains of Lb. delbrueckii
subsp. bulgaricus and S. thermophilus. NA-salicin medium did not give
good recovery of the commercial strain Lb. acidophilus La-145. It was
suggested that NA-salicin medium has the potential to differentiate
mixed probiotic cultures of bifidobacteria (NPNL), Lb. acidophilus
(MRS-clindamycin) and Lb. rhamnosus/Lb. paracasei in yoghurt
provided that strains are carefully selected (Van de Casteele et al.,
2006).
3.4. Media for enumerating Lb. casei
3.4.1. LC agar
There is a growing interest of adding Lb. casei as an adjunct
bacterium to yoghurt and probiotic cultures since Lb. casei is found to
be relatively stable than other yoghurt culture bacteria such as Lb.
acidophilus or Bifidobacterium (McCann et al., 1996; Shah et al., 1995).
If Lb. casei is also present in dairy products, then selective or dif-
ferential enumeration is complicated due to the lack of recovery of
one or more species, selectivity and/or differentiation ability amongst
colonies. However, there is little information on selective enumera-
tion of Lb. casei in yoghurt and fermented milk drinks, which may
contain yoghurt starter organisms and probiotic bacteria.
Selective enumeration of Lb. casei in yoghurt-type fermented milks
containing probiotic bacteria based on 15 °C incubation temperature
and 14 days incubation time was reported by Champagne et al.
(1997). However, an incubation time of 14 days may not be practical
for the dairy industry if the results are required in a short time. A
selective medium known as Lb. casei (LC) agar has been developed by
Ravula and Shah (1998) for enumeration of Lb. casei populations from
commercial yoghurts and fermented milk drinks that may contain
yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp.
bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. LC agar
inhibited the growth of S. thermophilus, Lb. delbrueckii subsp.
bulgaricus, Lb. acidophilus and bifidobacteria, and thus the medium
could be used to selectively enumerate Lb. casei.
In another study by Tharmaraj and Shah (2003), Lb. casei grew in
MRS-NaCl (4%), MRS-LiCl (0.5%) at 37 °C under anaerobic incubation
and LC agar. Lb. casei did not grow in a medium comprised of nalidixic
acid, paromomycin sulphate, neomycin sulphate and lithium chloride
and (NPNL) agar and at 43 °C however, lower incubation temperatures
(≤37 °C) supported the growth of Lb. casei. MRS-V agar at 37 °C or LC
agar at 27 °C under anaerobic incubation was found selective for Lb. casei
when Lb. rhamnosus was not present in a product. However, if Lb.
rhamnosus was present, total counts of Lb. casei and Lb. rhamnosus could
be obtained using MRS-V agar at 37 °C and anaerobic incubation for
72 h. To obtain the counts of Lb. casei, the count of Lb. rhamnosus on
MRS-V agar incubation at 43 °C for 72 h under anaerobic condition could
be subtracted from the total counts of Lb. casei and Lb. rhamnosus.
Talwalkar and Kailasapathy (2004) studied the growth of Lb. casei
on MRS-NPNL and predicted that the bigger colonies seen on MRS-NPNL
were those of Lb. casei. However, the counts of Lb. casei enumerated on
MRSB, MRS-LP or MRS-NPNL and those obtained on LC agar were found
inconsistent. The authors corroborated that LC agar offers good
selectivity and reliability of Lb. casei count in all the yoghurts on
which it was tested.
3.4.2. MRS-based agar
3.4.2.1. MRS-LP agar. For enumerating Lb. casei, the best media and
incubation conditions proposed by Lima et al. (2009) are MRS agar
with 2 g/L of lithium chloride and 3 g/L of sodium propionate (MRS-
LP) incubated at 37 °C or 42 °C, under aerobic or anaerobic conditions.
For enumeration of Lb. casei in a mixed culture, the media and
incubation conditions recommended were MRS-LP at 42 °C under
aerobiosis or anaerobiosis. With additional plating on MRS (or MRS
5.4) agar will lead to counts of S. thermophilus and Lb. delbrueckii
subsp. bulgaricus, which corresponded to the difference in the counts
in the two media. Vinderola and Reinheimer (2000) indicated that
differential enumeration of Lb. casei could be performed on MRS-LP
agar when it appears with Bifidobacterium. Lb. casei yielded round
white creamy colonies on media with diameter ranging from 1.7 to
2.4 mm and bifidobacteria yielded small round colonies ranging from
0.7 to 1.2 mm in diameter (Vinderola and Reinheimer, 2000).
3.4.2.2. MRSB agar. MRS agar containing 0.15% w/v of bile (MRSB) was
found useful in the selective colony count of Lb. acidophilus or Lb. casei
or in the selective and differential enumerations when these bacteria
appear together in a fermented dairy product (Vinderola and
Reinheimer, 2000). Lb. casei yielded round white creamy colonies
with diameter ranging from 0.9 to 1.3 mm; Lb. acidophilus appeared
on MRSB agar as irregular light brown colonies ranging in diameter
from 0.9 to 1.5 mm.
3.4.2.3. MRS-salicin agar. Lb. acidophilus and Lb. casei formed colonies
on MRS-salicin, MRS-sorbitol agar, MRS-ribose agar and MRS-
gluconate agar. Thus these media cannot be used for selective
enumeration of Lb. casei. MRS-salicin agar was reported to be selective
for Lb. acidophilus (Dave and Shah, 1996; Lankaputhra and Shah,
1996). However in another study, Lb. casei also grew in this medium
which affirms that MRS-salicin medium cannot be used for selective
enumeration of Lb. casei in products containing Lb. acidophilus and Lb.
casei (Ravula and Shah, 1998).
3.5. Media for enumerating Bifidobacterium
3.5.1. NPNL composite agar
3.5.1.1. NPNL agar. In order to improve the selectivity of media for
enumerating bifidobacteria, antibiotics, bifidogenic sugars or other
ingredients have been used: kanamycin, neomycin, paromomycin,
sodium propionate, lithium chloride, sorbic acid and sodium azide.
NPNL agar was developed by Teraguchi et al. (1978) for the
enumeration of bifidobacteria in dairy products containing mixtures
of bifidobacteria, lactobacilli and streptococci. The medium contain
BL-agar with neomycin sulphate, paromomycin sulphate, nalidixic
acid and lithium chloride and was then stated in more detail by Laroia
and Martin (1991). Bifidobacteria grow well in NPNL agar therefore it
is usually used as reference medium for the isolation of bifidobacteria
from fermented dairy products. Neomycin sulphate and nalidixic
acid were included as selective agents to inhibit growth of Gram-
positive and Gram-negative rods, respectively. Lithium chloride is a
substance commonly used for the selective isolation of bifidobacteria.
Although lithium chloride is commonly used in microbiology, still its
mechanism of action on bacterial cells is poorly understood. It is
suggested that low concentration of sodium salt (lithium chloride)
further prevents the growth of starter bacteria in yoghurt and in
cheese (Shah, 2000). Teraguchi et al. (1978) found that lithium
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
chloride at a concentration of 0.3% w/v restricted the growth of
Lb. acidophilus and had little effect on the growth of B. bifidum. Usually,
L-cysteine (0.05 to 0.1%) is also added to the media used for
enumeration of bifidobacteria to improve their recovery (Shah, 2000).
3.5.1.2. MRS-NPNL agar. NPNL added to several basal media (MRS or
RCA or TOS or RMS) has been attempted by many researchers to
selectively enumerate Bifidobacterium and to inhibit the growth of
other probiotic and yoghurt or cheese starter culture. MRS-NPNL
medium was found suitable for selective enumeration of bifidobac-
teria (Ravula and Shah, 1998). However they found poor recovery of
B. adolescentis and B. pseudolongum strains in this medium, suggesting
that counterchecking of pure strains of Bifidobacterium in this medium
is recommended before adopting them for enumeration purposes in
the presence of yoghurt cultures and Lb. acidophilus (Dave and Shah,
1996). Similarly, insufficient selectivity of MRS-NPNL was found
towards Lb. acidophilus and Lb. rhamnosus (strain LBA) by Van de
Casteele et al. (2006).
MRS-NPNL agar (which contains 0.05% of L-cysteine in the formula)
at 37 °C under anaerobic incubation supported the growth of only
bifidobacteria, so it is established that Bifidobacterium could be
enumerated on MRS-NPNL agar with 0.05% of L-cysteine under
anaerobic incubation at 37 °C for 72 h (Tharmaraj and Shah, 2003).
When L-cysteine was not present in the media, bifidobacteria either did
not grow or formed pinpoint colonies which depicts that subtracting L-
cysteine from other media could be used to control the growth of
bifidobacteria. As far as antibiotic resistance is concerned, Bifidobacter-
ium is relatively resistant to gentamicin, nalidixic acid and neomycin.
Conversely, starter lactococci, Lb. casei and Lb. acidophilus, are sensitive
to these antibiotics (Tharmaraj and Shah, 2003), so these antibiotics
could be used as growth controlling agent for these cultures, would help
to improve selectivity of media for bifidobacteria.
Talwalkar and Kailasapathy (2004) compared different media and
corroborate that MRS-NPNL and AMC agars were better for enumer-
ating bifidobacteria from probiotic yoghurts than MRS-LP or DP. In
another study, Van de Casteele et al. (2006) found NPNL and MRS-
LP media suitable for selective enumeration of the commercial
probiotic Bifidobacterium strains in yoghurt and cheese in combina-
tion with the tested starter cultures. Selective recovery of the tested
Bifidobacterium spp. was not uniform but resulted strain-to-strain
variations depending on whether NPNL or MRS-LP was used. The
preparation of NPNL was considered to be time-consuming but it
designated to be most selective medium used in the study.
3.5.1.3. TOS-NPNL agar. The addition of mixture of four antibiotics in
the same concentration as used in NPNL agar to trans-galactosylated
oligosaccharide (TOS) medium (TOS-NPNL) was proposed by Wijsman
et al. (1989) for the selective enumeration study of bifidobacteria. TOS-
NPNL agar inhibited the colony formation of bifidobacteria completely,
whereas an addition of the antibiotic mixture at a relative concentration
of 30% NPNL had no effect on the colony count of bifidobacteria
but inhibited the growth of pinpoint colonies of the streptococci
illustrating that the NPNL had no significant negative effect on the
recovery of colonies of bifidobacteria in the medium.
3.5.1.4. RMS-PPNL agar. Modified Rogosa agar with sodium propio-
nate, paromomycin sulphate, neomycin sulphate and lithium chloride
(RMS-PPNL) as compared to trypticase phytone yeast agar containing
neomycin sulphate, nalidixic acid and lithium chloride (TPY-NNL)
gave the highest recovery of bifidobacteria without any growth of
lactobacilli and streptococci (Samona and Robinson, 1991). The
selectivity of medium was checked using a mixture of yoghurt culture
(Lb. bulgaricus:S. thermophilus — 1:1) in the ratio of B. bifidum:yoghurt
culture — 1:1. The authors suggested that the medium could be
employed to monitor the survival of bifidobacteria in a range of dairy
products, and in the presence of other lactic acid bacteria. RMS-PPNL
203
was found inhibitory for B. bifidum and B. animalis (Ghoddusi and
Robinson, 1996). The same pattern was repeated on TPY with NPNL
and PPNL. The use of a lower concentration of NPNL gave good
recovery for both bifidobacteria whilst at the same time the growth of
cheese and yoghurt cultures was inhibited. These authors concluded
that the concentration of antibiotic should be carefully adjusted to
enumerate the precise strains being employed for any given product.
3.5.2. MRS-based agar
3.5.2.1. MRS agar. MRS agar is the media of choice for the enumeration
of lactic acid bacteria and probiotic organisms in cultured dairy
products, and is effective when Bifidobacterium is the only organism
present. Selectivity of MRS can be enhanced by addition of specific
agents such as cycloheximide (antibiotic inhibiting growth of yeasts)
or sorbic acid (inhibiting growth of yeast by reduction of pH of
medium from 6.2 to 5.7) (Reuter, 1985). Dave and Shah (1996)
suggested an excellent recovery of Bifidobacterium found on MRS agar.
More frequently, however, inhibitors or supplements are used in
combination with other compounds to enhance selectivity of the
bifidobacteria and inhibit the growth of the lactic acid bacteria.
3.5.2.2. BSM. MRS medium supplemented with cysteine hydrochloride
0.5 g/L and mupirocin 50 mg/L to form Bifidobacterium selective
medium (BSM) was found to be elective for Bifidobacterium species
and selective in the presence of bacilli, lactococci, lactobacilli and
streptococci commonly found in animal feed. In addition, enterococci,
pediococci and propionibacteria formed colonies N0.5 mm that were
readily distinguished from bifidobacteria colonies that were ≥1 mm
in diameter (Simpson et al., 2004). There are, however, ethical issues
concerning the use of the antibiotic “mupirocin” which has particular
significance in the treatment of MRSA (methicillin resistant Staphy-
lococcus aureus) and H. pylori infection. Because of concerns about the
emergence of mupirocin-resistant MRSA, it is far-sighted by some
authors to expect difficulty in obtaining this antibiotic in future
(Upton et al., 2003). However the selective complement of mupirocin-
supplement (89914, Sigma-Aldrich) and mupirocin (M7694, Sigma-
Aldrich) are readily available but mupirocin has been found
comparatively expensive than other antibiotics such as dicloxacillin.
3.5.2.3. MRS-raffinose agar. The selective enumeration of B. lactis BB12
was obtained using MRS-raffinose at 45 °C for 72 h under anaerobic
conditions. The medium comprised of MRS fermentation broth
supplemented with raffinose (1%), LiCl (0.05%), cysteine (0.05%)
and agar (1.5%). Selective conditions for enumeration of this species
were incubation at 45 °C, use of raffinose as a carbohydrate source and
addition of LiCl to suppress lactobacilli growth. The method was
suggested to be selective for B. lactis against the LAB strains studied
(Tabasco et al., 2007).
3.5.2.4. MRSD agar. Lima et al. (2009) compared the growth capability
of probiotic and non-probiotic cultures on twenty-one culture media
and reported MRS agar with dicloxacillin incubation at 37 °C or 42 °C
under anaerobiosis as the best culture media and best incubation
conditions for the enumeration of B. animalis. However for enumer-
ation of B. animalis in a mixed culture, the recommended media and
incubation conditions includes MRS agar added with dicloxacillin
(2 mg/L), lithium chloride (1.1 g/L) and cysteine (0.5 g/L) at 42 °C and
anaerobiosis. Lima et al. (2009) found that B. animalis Bb12 grew in
MRS agar added with maltose (MRSM) but not in MRS agar added
with trehalose (MRST) confirming results reported by Vinderola and
Reinheimer (1999). Dicloxacillin present in MRS agar inhibited the
growth of yoghurt bacteria without affecting the growth of bifido-
bacteria. Higher concentration (2 mg/L) of dicloxacillin was found
inhibitory for the two lactobacilli strains (Lb. acidophilus La05 and Lb.
casei Lc01). Gentamycin also affected the two strains in a different
204 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
way such as when the plates were incubated at 37 °C under aerobiosis
or anaerobiosis this antibiotic presented no inhibitory effect.
However, if incubated at 42 °C, Lb. casei Lc01 was inhibited but not
Lb. acidophilus La05 (Lima et al., 2009). It was concluded that MRS
with dicloxacillin can be used for enumeration of B. animalis Bb12 in
the presence of dairy cultures (S. thermophilus and Lb. delbrueckii
subsp. bulgaricus) or the other two probiotic microorganisms tested.
The incubation temperature can be either 37 °C or 42 °C, but the
atmosphere should be anaerobic.
3.5.2.5. MRSM agar. In another study by Rybka and Kailasapathy
(1996), higher recovery of bifidobacteria (mixture of B. bifidum and B.
longum) from experimental yoghurt was found on MRSM agar as
compared with RCPB 5.0, suggesting the suitability of MRSM for
enumerating bifidobacteria compared with the later media.
3.5.3. BLOG agar
Some of the researchers found difficult to prepare NPNL because it is
time consuming to weigh 24 ingredients and to filter sterilise few of
them. They focused on developing selective media using commercial
ones containing simpler composition than NPNL. Lim et al. (1995)
proposed blood-glucose-liver agar with oxgall and gentamycin (BLOG),
a selective medium for bifidobacteria in fermented dairy products. The
authors found it much simpler to prepare and most suitable in giving
higher recovery of bifidobacteria in yoghurts than NPNL agar. The
medium gave 90% or higher recovery of bifidobacteria compared with
that on BL agar expect for one strain of B. longum. The media comprised
BL agar containing oxgall (0.2 mg/mL) and gentamycin (30 μg/mL) as
selective agents. Oxgall was found to be inhibitory to other non-
intestinal lactic acid bacteria at 0.2 mg/mL concentration and gentamy-
cin was found to have optimal selectivity for bifidobacteria at 30 μg/mL
concentration. All tested strains of Lb. acidophilus, Lb. delbrueckii subsp.
bulgaricus and Lb. casei were inhibited in BLOG and NPNL. Pin-pointed
colonies of one strain of S. thermophilus were found in NPNL whereas all
strains of streptococci were inhibited in BLOG suggesting, the NPNL agar
had fewer inhibitory effects on S. thermophilus strains. BLOG also
showed good inhibition to yoghurt culture and Lb. acidophilus. However,
Payne et al. (1999) found that higher percentage recoveries with NPNL
than BLOG, for B. longum by spread and pour plate and of B. bifidum by
spread plate. They concluded, as for NPNL, the major disadvantage of
this medium is that it is time-consuming to prepare. As far as plating
technique is considered, in contrast with previous report, Lima et al.
(2009) compared the growth capability of probiotic and non-probiotic
bacteria on group of twenty-one culture media and reported no
significant differences (P ≤ 0.05) in counts using pour plating or surface
plating in all experiments.
Antunes et al. (2007) used specific culturing conditions for selective
enumeration study of B. animalis subsp. lactis in buttermilk product
aiming at inhibiting mesophilic aromatic cultures (MAC) composed of
multiple mixed strains including Lc. lactis subsp. cremoris, Lc. lactis
subsp. lactis, Lc. lactis subsp. lactis biovar. diacetylactis and Leuconostoc
mesenteroides subsp. cremoris. These culturing conditions include
incubation temperature above 37 °C or pH 4.8 for Leu. mesenteroides
inhibition and temperature of 45 °C or 4% NaCl for Lactococcus ssp.
inhibition. MRS agar supplemented with 0.5% L-cysteine HCL at 10%,
1% lithium chloride at 10% and 0.02% aniline blue were tested with
gradient addition of 0.5% dicloxacillin. The medium variation using
different antibiotic concentrations, addition of NaCl and raising pH
was not found to be remarkable; however, thermophilic incubation
was concluded to be an efficient growth parameter for the selective
enumeration of B. animalis subsp. lactis Bb12 in the presence of MAC.
3.5.4. CAB medium
3.5.4.1. MCAP medium. Modified Colombia agar base supplemented
with propionic acid (MCAP) at pH 5.0 was described as selective and
elective by Beerens (1991). Silvi et al. (1996) reported MCAP agar the
most selective amongst three media studied, inhibiting the growth of
six out of nine non-bifidobacteria strains tested, whilst at the same time
allowing enumeration of four out of five Bifidobacterium strains tested.
For two strains of bifidobacteria it was the medium yielding the closest
viable counts to those on the control medium. It was anticipated the
most suitable medium for isolation and enumeration of Bifidobacterium
spp. from the gut microflora (Silvi et al., 1996). In contrast of previous
report, Payne et al. (1999) stated that MCAP medium was not found as
inhibitory to the growth of Lb. bulgaricus, S. thermophilus and Lb.
acidophilus as the other selective media assessed because it gave
comparatively poor recovery of B. longum and no growth of B. bifidum.
3.5.4.2. CAB-NL medium. Chapon and Kiss (1991) proposed Columbia
agar base (CAB) medium with neomycin sulphate and lithium
chloride (CAB-NL) more selective than the medium proposed by
Beerens (1990). Roy et al. (1997) initially found that viable counts of
bifidobacteria on modified Columbia agar base (MCAB) with 0.05%
cysteine–HCl plus raffinose (0.5%) containing various selective agents
(LP, NPNL and gentamycin) were comparable to those on non-
selective media such as modified MRS (MMRS).
3.5.4.3. RAF 5.1 medium. The MCAB plus raffinose medium supple-
mented with lithium chloride (2 g/L) and sodium propionate (3 g/L)
with pH adjusted to 5.1 (RAF 5.1) was used successfully as a selective
medium for the enumeration of Bifidobacterium from fresh cheese (Roy
et al., 1997). Viable count of bifidobacteria was comparable to those on
MCAB plus LP. The gentamycin or NPNL solution was added to MCAB
plus raffinose to replace LP mixture. MCAB plus raffinose containing
gentamycin or NPNL were named as RAF-G and RAF-NPNL respectively.
These modified raffinose media could be used for manufacture of fresh
cheese as raffinose is not metabolised by lactococci. The authors found
that Bifidobacterium could survive up to 15 days at a level higher than
106 cfu/g in a fresh cheese stored at 4 or 12 °C however, the decrease in
the viable count was faster during storage at 4 °C than at 12 °C. They
observed that the growth of S. thermophilus, Lb. acidophilus and Lb.
bulgaricus strains was inhibited on the MCAP plus raffinose medium.
The authors recommended RAF 5.1 because it is simple and easy to
prepare than RAF-G or RAF-NPNL.
3.5.4.4. MCAB-LG medium. Roy et al. (1997) recommended the use of
MCAB plus lactose and gentamycin, for the selective enumeration of B.
bifidum because some strains of this species are raffinose negative.
3.5.4.5. DP medium. DP medium-combination of Columbia agar
supplemented with propionic acid (5 mL) and dicloxacillin (2 mg/
L), pH adjusted to 6.8, allowed the growth of bifidobacteria and
prevented the growth of Lb. acidophilus, S. thermophilus and Lb.
delbrueckii subsp. bulgaricus (2001). The authors recommended the
medium for the selective isolation of bifidobacteria from starter
cultures and from different fermented milks products. It was
confirmed in another report that DP agar inhibited the growth of Lb.
acidophilus and other yoghurt starter cultures (Roy et al., 1997).
3.5.5. RCA-based medium
3.5.5.1. RCA. For routine enumeration of Bifidobacterium from pure
cultures, commercially available non-selective media such as RCA and
MMRS prove to be time and cost effective in addition of giving excellent
recovery of bifidobacteria. Dave and Shah (1996) found that the recovery
of all bifidobacteria obtained on RCA was almost similar to that on MMRS
at pH 6.8, but the recovery was slightly lower on RCA at pH 5.3.
3.5.5.2. BIM25. Munoa and Pares (1988) developed a selective
medium called Bifidobacterium iodoacetate medium 25 (BIM25), for
isolation and enumeration of Bifidobacterium spp. from water
 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
samples. RCA supplemented with nalidixic acid, polymixin B sulphate
and kanamycin sulphate constituted RCA17 medium. BIM25 medium
was prepared by adding iodoacetic acid (25 mg/L) and 2,3,5-
triphenyltetrazolium chloride (TTC: 25 mg/L) into RCA17 as a basal
medium. Iodoacetate inhibits glyceraldehyde-3-phosphate dehydro-
genase and reduces the growth of non-bifidobacterial contaminant
colonies. BIM25 was found to be more selective than RCA17 tested in
the study however it was also somewhat toxic to some of the
bifidobacteria. In experiments carried out with pure cultures of 12
Bifidobacterium strains, BIM25 inhibited only two strains of B.
adolescentis. These two strains grew well if the concentration of
iodoacetate was reduced by half, but this resulted in a decrease of
selectivity. From the experiments carried out with several Bifidobac-
terium spp., Munoa and Pares (1988) concluded that, when stressed
by adverse environmental conditions, the organisms may become
unable to grow on BIM25 medium and the problem was overcome by
incorporating resuscitative incubation on RCA into the enumeration
procedure. Silvi et al. (1996) also found BIM25 very selective medium,
inhibiting six of the nine non-bifidobacteria strains tested. On the
other hand it also inhibited the growth of Bifidobacterium strains with
pure cultures grown on modified BIM25 (MBIM25) showing consis-
tently lower cfu/mL values than on the control medium.
For the enumeration of B. infantis or B. lactis in the nutritional
products, modified Bifidobacterium iodoacetate medium (MBIM:
12.5 mg iodoacetic acid/L) was compared to MRS agar supplemented
with bile, cysteine, and dicloxacillin (MRS-BCD). Both of the two
media performed similarly for enumerating B. infantis in product
stored at 22 °C in sealed cans. However, weak correlation (r 2 b 0.50)
was found between MBIM and MRS-BCD for enumerating B. infantis
and B. lactis in products stored in sealed cans at high temperature or in
open cans at high relative humidity. It was concluded that the MBIM
medium cannot be considered as a sole medium for enumeration of
probiotic Bifidobacterium spp. in powdered nutritional products
stored under high temperature and/or high relative humidity
conditions (Ingham, 1999).
3.5.6. LP composite agar
3.5.6.1. LP agar. Lapierre et al. (1992) formulated lithium chloride–
sodium propionate (LP) agar for selective enumeration of Bifidobac-
terium from commercial products by combining lithium chloride (2 g/
L) and sodium propionate (3 g/L) to liver-cystine-lactose (LCL) agar to
suppress the growth of lactic acid bacteria. Sodium propionate was
used as a selective agent in the medium. LP was found to give similar
enumeration results as found on NPNL agar used as reference medium
and hence it was given more importance than NPNL in terms of ease of
preparation and short incubation requirement. However, in one of the
study by Payne et al. (1999) LP proved to be a poor medium for
selective enumeration of the strains of B. longum and B. adolescentis
but LP agar proved to be suitable for the selective enumeration of B.
bifidum at 37 °C by the spread-plate method. The authors found that
LP did not prove to be sufficiently selective against a yoghurt culture
incubated at 40 °C, as percentage recovery was 20.9% and Lb.
acidophilus did not grow on LP at either 37 °C or 40 °C for spread or
pour plates.
3.5.6.2. AMC agar. Arroyo, Martin and Cotton (AMC) agar was
developed by Arroyo et al. (1995) by adding the selective components
in LP agar into modified BIM25 (MBIM-1) agar. The BIM 25 was
modified by either reducing the iodoacetate concentration by one half
(MBIM-1) or by completely excluding iodoacetate (MBIM-2). The
growth of Lb. acidophilus was found on MBIM-1 whereas it was
completely inhibited on LP agar. AMC agar supported the growth of
Bifidobacterium whilst inhibiting the growth of Lb. delbrueckii subsp.
bulgaricus and S. thermophilus. It was however, found to allow slight
growth of Lb. acidophilus (Arroyo et al., 1995; Payne et al., 1999).
205
AMC medium comprised of reconstituted clostridial agar medium
(3.8%), nalidixic acid (2%), sodium propionate (0.3%), lithium chloride
(0.2%), and agar (1.3%) with a filter-sterilised antibiotic solution
containing polymyxcin B sulphate (8.5 mg), kanamycin sulphate
(50 mg), iodoacetic acid (12.5 mg), and 2,3,4-triphenyltetrazolin-C
(125 mg).
Corbo et al. (2001) compared the efficacy of MMRS, RMS, NPNL
and AMC for selective enumeration of bifidobacteria in dairy products
by using spread- and pour-plate techniques. In their study, MMRS and
AMC media and pour-plate technique gave the highest cell recovery of
B. bifidum Bb02 and B. longum Bb46 grown in milk with differences of
ca. 2 log10 cfu/g compared to the other media. In contrast with other
reports (Payne et al., 1999; Roy et al., 1997) the starter lactic acid
bacteria i.e. Lb. delbrueckii subsp. bulgaricus and S. thermophilus, used
in the production of Canestrato Pugliese cheese, were also recovered
on MMRS and AMC media, although at cell numbers two orders of
magnitude lower than those determined in MRS medium. The
recovery of bifidobacteria on AMC medium was slightly lower than
that on MMRS medium (Corbo et al., 2001).
AMC agar was found to be most appropriate media for the selective
enumeration of bifidobacteria from mixed culture, in quite few
reports reviewed (Arroyo et al., 1995; Darukaradhya et al., 2006;
Payne et al., 1999). AMC, RMS, NPNL and BLOG were evaluated by
Payne et al. (1999) as selective media for bifidobacteria and all of
these media gave good recoveries of bifidobacteria and inhibited the
growth of Lb. delbrueckii subsp. bulgaricus, S. thermophilus and Lb.
acidophilus. However, the authors concluded that out of these four
media, AMC was most convenient as it is a commercially available
medium whereas the others must be made up from individual
constituents and it offers a good choice for the routine enumeration of
bifidobacteria from mixed cultures.
Sanders et al. (1996) tested AMC agar on five strains of
bifidobacteria after finding LP and M17 containing lactose and lithium
chloride (M17LL) ideally unsuitable. All strains showed statistically
significant reductions in counts compared with counts on reinforced
clostridial agar (RCA) as non-selective medium. However, two strains
appeared most dramatically inhibited. This observation led Sanders et
al. (1996) to suggest that the AMC medium is not an optimal medium
for differential enumeration at least for commercial strains used in the
study. All lactobacilli and most bifidobacteria tested grew on agar
plates containing up to 3% bile whereas all streptococci were sensitive
to the lowest concentrations of bile tested. The authors concluded that
additional research in this area is required because several bifido-
bacteria showed significant differences between the selective and
non-selective media. With reference to the controversial and contrary
reports against AMC, the medium leaves doubt to be used as a
selective medium and needs to be evaluated in this regard.
3.5.6.3. GL agar. Iwana et al. (1993) prepared galactose agar (GL) for
isolation and identification of Bifidobacterium spp. in commercial
yoghurts sold in Europe. The medium contains lithium chloride
(0.4 g/L) and galactose (10 g/L) as selective inhibitory agents. In
order to measure the ability to recover Bifidobacterium spp. from pure
cultures comparison was made between GL agar and previously
mentioned BL agar. All the tested strains of Bifidobacterium grew well
and gave almost same count on both media. Except for Lb. helveticus
and Lb. casei, no strains of the facultative aerobes, were detected on
GL agar. In the light of results obtained, GL agar was proposed for the
selective enumeration of bifidobacteria with addition of lithium
chloride in lower concentrations than that of BL agar supplemented
with lithium chloride, paromomycin sulphate, neomycin sulphate
and sodium propionate.
3.5.6.4. RB agar. Raffinose-Bifidobacterium (RB) agar — a new selective
medium for the detection of bifidobacteria developed by Hartemink
et al. (1996) offers suitability in terms of lack of antibiotics and ease of
206 R. Ashraf, N.P. Shah / International Journal of Food Microbiology 149 (2011) 194–208
preparation. Selectivity of the medium based on the presence of
propionate (15 g/L) and lithium chloride (3 g/L) as inhibitory agents,
and raffinose (7.5 g/L) as a selective carbon source. Casein (5 g/L) was
incorporated as a protein source to facilitate the formation of zone of
precipitation around the yellow colonies with a yellow halo. All dairy
bifidobacteria found to grew well on RB agar, except for some
B. bifidum strains. However, distinctive growth characteristics were
not found for non-bifidobacterial strains used in dairy products.
Hartemink et al. (1996) suggested RB agar to be more selective than
the other media compared and used.
3.5.6.5. BFM. Nebra and Blanch (1999) proposed another antibiotic-
free selective medium called Bifidobacterium medium (BFM) which
includes methylene blue, propionic acid, and lithium chloride as
inhibitors of some related bacterial species and lactulose as a main
carbon source for Bifidobacterium. The low pH (5.5) of the medium
contributes to the inhibition of the growth of Enterobacteriaceae. In
order to measure the recovery on BFM agar, two media were used for
comparison: Columbia blood agar (CBA) supplemented with glucose
and L-cysteine as an enriched medium and a Bifidobacterium medium
includes BL medium. Four tested isolates that do not belong to the
Bifidobacterium genus grew on both media. However, these isolates
were easily differentiated from Bifidobacterium spp. because of their
small diameters (b0.5 mm) and differential colonial morphologies.
Similar colony counts were obtained on BFM, CBA and glucose blood
liver (BL) medium for most of the 26 Bifidobacterium strains tested.
Nebra and Blanch (1999) verdict that the simple composition and
high level of recovery BFM medium yielded makes it a feasible for
routine analysis and routine enumeration of Bifidobacterium in
fermented dairy products such as yoghurts.
3.5.7. TPY-based agar
3.5.7.1. MTPY agar. Modified trypticase phytone yeast extract (MTPY)
agar (modified by the addition of mupirocin 100 mg/L) was claimed
to be highly selective and suitable for isolation and enumeration of
bifidobacteria in dairy products (Vlková et al., 2004). The study by
Samona and Robinson (1991) favours the previous report as TPY
medium also gave high counts of B. bifidum and offers suitability for
the selective isolation and growth of bifidobacteria. In addition, the
authors stated that the RMS-PPNL (4.0 mL of PPNL/100 mL medium)
was the best selective medium which gave the highest counts of
B. bifidum without any growth of lactobacilli or streptococci (Samona
and Robinson, 1991).
3.5.7.2. TPYD agar. Sozzi et al. (1990) used the antibiotic dicloxacillin
for isolating and counting the Bifidobacterium present in fermented
milks. The addition of 2 μg/mL of dicloxacillin to TPY medium (TPYD)
was found inhibitory to the growth of lactobacilli and streptococci
whereas the medium supported the growth of most Bifidobacterium
very well. TPYD medium was found to be more suitable than MRS agar
added with dicloxacillin (MRSD) to select Bifidobacterium. The authors
concluded that ‘the addition of dicloxacillin at a concentration of 2 μg/
mL to MRS or, better still, to TPY media can most certainly be
recommended for Bifidobacterium isolation and count in fermented
milks or dairy products in general’ (Sozzi et al., 1990).
3.5.8. WCM agar
Rada and Koc (2000) proposed Wilkins-Chalgren agar containing
1% w/v mupirocin (WCM), for the isolation and enumeration of
bifidobacteria in cultured milk products. The study establishes that
strains of lactobacilli, lactococci, leuconostocs and streptococci were
completely inhibited on this selective medium, whereas recovery of
bifidobacteria was found to be about 100%. The total counts on
mupirocin agar were practically identical with those found on the
control WCM, whilst the NPNL medium gave significantly lower
numbers of bifidobacteria. In this regard, the proposed medium is
claimed to be selective for bifidobacteria in probiotics that contain
mixed population of lactic acid bacteria and bifidobacteria. Another
advantage of this medium as compared to others is its ease of
preparation.
In one of the study, the media MRS containing ox-bile (MRSO),
WCM and NPNL were found not to be selective for bifidobacteria since
they allowed growth of Lb. acidophilus strains and AMC was found to
inhibit the growth of Lb. acidophilus but the growth of starter lactic
acid bacteria (SLAB) or non-starter lactic acid bacteria (NSLAB) were
not inhibited (Darukaradhya et al., 2006).
4. Conclusion
Differential enumeration of yoghurt bacteria could be performed
on TPPY-based agar, MMRS-BPB agar, RCPB agar, Lee's agar, LB agar,
HHD agar or BGW agar. MRSF, MRS 5.2, RCPB 5.3 or RCA 5.3 are
suitable for Lb. delbrueckii subsp. bulgaricus when the incubation is
carried out at 45 °C for 72 h. Selective enumeration of S. thermophilus
could be carried out at 37 °C on ST agar and at 45 °C on M17L agar
under aerobic incubation. MRSM agar could be used to get the total
count of Lb. acidophilus and bifidobacteria. BAM agar under anaerobic
incubation at 43 °C, MRS-clindamycin at 37 °C or X-Glu agar could be
used for the selective enumeration of Lb. acidophilus in yoghurt-
related milk products containing mixed microflora of lactobacilli,
streptococci and bifidobacteria. Lb. casei could be selectively enumer-
ated on specially formulated LC agar from commercial yoghurts and
fermented milk drinks. Bifidobacterium could be enumerated on MRS-
NPNL agar added with 0.05% of L-cysteine under anaerobic incubation
at 37 °C for 72 h.
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