media for the isolation and enumeration of bifidobacteria in dairy products

Ž .International Journal of Food Microbiology 69 2001 167–182www.elsevier.comrlocaterijfoodmicro

Review

Media for the isolation and enumeration of bifidobacteria indairy products

Denis Roy)

Food Research and DeÕelopment Centre, Agriculture and Agri-food Canada, 3600 CasaÕant BlÕd. W., Saint Hyacinthe,QC, Canada J2S 8E3

Received 18 September 2000; received in revised form 12 February 2001; accepted 16 February 2001

Abstract

Bifidobacteria are commonly used for the production of fermented milks, alone or in combination with other lactic acidbacteria. Bifidobacteria populations in fermented milks should be over 106 bifidobacteriarg at the time of consumption ofstrain added to the product. Hence, rapid and reliable methods are needed to routinely determine the initial inoculum and toestimate the storage time period bifidobacteria remain viable. Plate count methods are still preferable for quality controlmeasurements in dairy products. It is, therefore, necessary to have a medium that selectively promotes the growth ofbifidobacteria, whereas other bacteria are suppressed. The present paper is an overview of media and methods includingsummaries of published comparisons between different selective media. Culture media for bifidobacteria may be divided intobasal, elective, differential and selective culture medium. Non-selective media are useful for routine enumeration of

Ž .bifidobacteria when present in non-fermented milks. Reinforced Clostridial Agar and De Man Rogosa Sharpe MRSsupplemented with cysteine and agar available commercially are the media of choice for industrial quality controllaboratories. Several media for selective or differential isolation have been described for enumeration of bifidobacteria fromother lactic acid bacteria. From the large number of selective media available, it can be concluded that there is no standardmedium for the detection of bifidobacteria. However, Columbia agar base media supplemented with lithium chloride andsodium propionate and MRS medium supplemented with neomycin, paromomycin, nalidixic acid and lithium chloride canbe recommended for selective enumeration of bifidobacteria in dairy products. q 2001 Elsevier Science B.V. All rightsreserved.

Keywords: Probiotics; Bifidobacteria; Selective; Elective; Basal; Media

1. Introduction

During the last three decades, attempts have beenmade to improve the health status of human bymodulating the intestinal microflora using live mi-

Žcrobial adjuncts called probiotics Holzapfel et al.,

) Fax: q1-450-773-8461.Ž .E-mail address: [email protected] D. Roy .

.1998 . Indeed, different products containing probi-otic bacteria have gained in popularity with con-sumers. The consumption of these microorganismsmay affect the composition of indigenous microfloraand may have several beneficial effects on the hu-man health such as the maintenance of a balancedflora, alleviation of lactose intolerance symptomsand resistance to enteric pathogens. Probiotics canalso prevent or ameliorate diarrhea through their

Žeffects on immune system De Roos and Katan,

0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0168-1605 01 00496-2

( )D. Royr International Journal of Food Microbiology 69 2001 167–182168

.2000 . However, it is important to note that anypostulated benefit from consumption of probioticsshould be accepted as facts only after extensive

Ž .testing in human clinical studies Rolfe, 2000 .The incorporation of new bacteria of intestinal

origin into human diet corresponds to the emergenceof a new generation of food products, which use thebeneficial effects of these bacteria on intestinalmetabolism. Recent advances of research in intesti-nal flora are the background for the appearance of

Ž .functional foods Mitsuoka, 2000 . Bifidobacteria areemerging as possibly one of the most importantgroup of intestinal organisms with regard to humanhealth. Yogurts with bifidobacteria have beenlaunched for decades in Europe and Japan and their

Ž .consumption is yet increasing Mitsuoka, 2000 . Bi-fidobacterium bifidum, B. breÕe, B. longum and B.animalis are commonly used for the production offermented milks, in combination with other lacticacid bacteria.

Tissier first described bifidobacteria in 1900.Ž .Tissier 1900 noted that bifidobacteria were the

dominant bacteria in the stools of breast-fed infantsand were more numerically significantly than in thestools of bottle-fed infants. They are Gram-positive,non-spore forming, strictly anaerobic and pleomor-phic fermentative rods, often Y-shaped. The GqCcontent of DNA varies from 55 to 67 mol%. Theoptimum growth temperature for bifidobacteria is

Ž378C to 418C minimum growth temperature: 25–.288C; maximum growth temperature: 43–458C and

Žthe optimum pH is 6.5 to 7.0 no growth at 4.5–5.0.or 8.0–8.5 . Glucose is degraded exclusively and

characteristically by the fructose-6-phosphate shunt.Fructose-6-phosphate phosphoketolase is the charac-teristic key enzyme of the bifid shunt that cleavesfructose-6-phosphate into acetyl phosphate and ery-throse-4-phosphate. Acetic and lactic acid are formed

Ž .primarily in the molar ratio of 3:2 Scardovi, 1986 .Thirty-three species of bifidobacteria are now in-

cluded in this genus and twelve of these species havebeen found in the intestine of man andror as humanclinical isolates. The remaining 21 species have beenisolated from fermented milks, the alimentary tractsof various animals and honeybees and found also in

Ž .sewage and anaerobic digesters Scardovi, 1986 .Bifidobacteria constitute 5% to 10% of the totalcolony flora of healthy children and adults. In the

days following birth, infants have intestinal floradominated by bifidobacteria. With age and changesin dietary habits, bifidobacteria tend to be suppressedby other microorganisms and their population even

Ž .decreases in humans, late in life Mitsuoka, 2000 .The main species present in humans are B. adoles-centis, B. bifidum, B. infantis, B. breÕe and B.longum in the colon. Their presence in the humanintestine is almost universally accepted to be a con-

Žtributing factor to a healthy well being O’Sullivan.and Kullen, 1998 . It has been claimed that ingestion

of specific bifidobacteria could contribute to reestab-lishment of a bifidobacterial flora in humans afterantibiotic therapy; alleviation of constipation; pre-vention against diarrhea and other gastrointestinalinfections and alleviation of the symptoms of lactose

Ž .intolerance O’Sullivan and Kullen, 1998 .Selection of probiotics can be based on general

microbiological criteria that refer to safety, technol-Žogy, performance and health benefits Gibson and

.Fuller, 2000 . High viable counts and survival ratesduring stomach passage are necessary to allow livebifidobacteria from the fermented milk products to

Žplay a biological role in the human intestine Reuter,.1990; Kneifel et al., 1993; Tamime et al., 1995 . The

technological aspects for selection of strains shouldinclude the following criteria: strains claimed to bepresent in a product should survive in relatively highviable cell numbers, retain metabolic activity and

Žprovide desirable organoleptic qualities Holzapfel et.al., 1998 . It is important that bifidobacteria survive

in fermented dairy products until consumption. Theviability of bifidobacteria depends on the degree ofacidification and on the bacterial strains, fermenta-tion conditions, storage temperature, and preserva-tion methods and is mainly limited by their sensitiv-

Ž .ity to the high acidity Shah, 1997 .In general, the food industry has targeted popula-

tions over 106 bifidobacteriarg at the time of con-Žsumption of strain added to food Samona and

.Robinson, 1991; Sanders et al., 1996 . This standardappears to have been adopted to provide bacterialconcentrations that were technologically attainableand cost-effective rather than to achieve a specific

Ž .health effect in humans Sanders et al., 1996 . Theneed exists for rapid, reliable methods for enumera-tion of bifidobacteria. Such methods are needed toroutinely determine the initial inoculum and to esti-

( )D. Royr International Journal of Food Microbiology 69 2001 167–182 169

mate the storage time period these organisms remainŽ .viable Chapon and Kiss, 1991; Arroyo et al., 1994 .

Ž .Charteris et al. 1997 stressed that quality assur-ances programmes in the research, development, pro-duction and validation of the health benefits of theprobiotic products require microbiological proce-dures for the detection, identification and differentialenumeration of probiotic microorganisms. Severalmethods could be applied for the detection of bifi-dobacteria in dairy products, like plate count meth-ods, DNA-based methods or enzymatic methods.

Ž .According to Hartemink et al. 1996 , plate countmethods are still preferable for quality control mea-surements in dairy products. It is, therefore, neces-sary to have a medium that selectively promotes thegrowth of bifidobacteria, whereas other bacteria aresuppressed.

Various media have been proposed and used overthe past 60 years for the isolation, cultivation andenumeration of bifidobacteria from the intestinal tract

Ž .or faeces of human Rasic and Kurmann, 1983 andwill not been reviewed because these methods havebeen extensively described by Rasic and KurmannŽ .1983 . Comprehensive reviews of culture media fordetection and enumeration of bifidobacteria in fer-mented milks have also been published by Rasic and

Ž . Ž .Sad 1990 and by Tamime et al. 1995 . Charteris etŽ .al. 1997 reviewed selective and differential plating

methodologies used for isolation, identification andenumeration of potentially probiotic lactobacilli andbifidobacteria. They described differential platingmethodologies for detection and enumeration ofLactobacillus acidophilus and Bifidobacteriumspecies alone or in combination with yoghurt starter

Ž .bacteria. Charteris et al. 1997 also discussed theuse of a large number of nucleic acid methodsdeveloped for the specific detection of lactobacilliand bifidobacteria. The present paper is an overviewof media and methods including summaries of pub-lished comparisons between different selective me-dia.

Ž .Rasic and Sad 1990 indicated that two factorsare important in detecting and enumerating bifi-dobacteria. These factors are an adequate culturemedium and anaerobic conditions. With new tech-nology now available for providing anaerobic condi-tions, bifidobacteria are now being studied easily and

Ž .more extensively Laroia and Martin, 1991 . The

success of bifidobacteria detection in an optimalculture medium is mainly dependent upon the fol-

Ž .lowing factors: a If the culture medium has noselective effect, non-bifidobacteria may outgrow bifi-

Ž .dobacteria; b the macroscopic identification of bifi-dobacteria colonies may be facilitated using differen-

Ž .tial coloring; c the optimal growth of bifidobacteriais dependent upon the freshness of the ingredients of

Ž .the medium; and d the composition of the culturemedium allow the growth of different biotypes pre-

Žsent in the investigated material Rasic and Kur-.mann, 1983 .

Ž .According to Rasic and Kurmann 1983 , culturemedia for bifidobacteria may be divided into com-plex, selective, semisynthetic, synthetic as well ascommercial culture medium. The following parame-ters should be considered for the selection of anadequate culture medium for bifidobacteria: supplyof nutritive and growth substances; low oxidation–reduction potential; maintenance of the pH valueduring growth and the buffering capacity; final pH ofthe prepared medium; optimal growth media.

Ž .Hartemink and Rombouts 1999 indicated thatmedia used for the detection of bifidobacteria can beclassified into five different groups: known non-

Ž .selective media MRS, Rogosa , media with electivecarbohydrates, media with antibiotics, media withpropionate and media with elective substance androrlow pH. Combinations, media belonging to morethan one group, are also used. From the large num-ber of media used, it can be concluded that there isno standard medium for the detection of bifidobacte-ria.

The availability of easy and inexpensive methodsfor detection, identification, and enumeration of Bifi-dobacterium is consequently important in food

Ž .microbiology Nebra and Blanch, 1999 . Several me-dia for selective or differential isolation have beendescribed. Most of them have complex compositionsthat include antibiotics as growth inhibitors, requirelong incubation times, or show low bacterial recov-ery levels.

2. Sample plating

Preparation of samples for dilution plates mayconsist of manually shaking with a known volume of


diluent or macerated with a diluent in a stomacher.Ž .Peptone 0.1% water and peptone water with saline

Ž .0.85% are among the most commonly used diluentsfor enumeration of bifidobacteria in dairy productsŽ . Ž .Table 1 . Collins and Hall 1984 tested the follow-ing diluents for use in enumerating cultures of bifi-dobacteria: 0.9% saline, 0.1% peptone, 0.9% salineplus 0.1% peptone, and saline plus peptone bufferedwith 0.03% KH PO plus 0.06% Na HPO . No2 4 2 4

significant difference was obtained except with pep-tone alone, the counts were about 70% of those with

Ž .the other diluents. Hartemink and Rombouts 1999tested the effect of dilution medium such as physio-

Ž .logical salt solution ps and reduced physiologicalŽsalt solution ps with 0.5 grl cysteine; HCl, pH 6.7

on the total plate counts of faecal samples. Theyfound that physiological salt and reduced physio-logical salt gave very good results. The effect ofdiluting under aerobic and anaerobic conditions wastested for these dilution solutions and no significantdifferences were observed between diluting and plat-ing the samples aerobically or anaerobically. Theyobserved that viable counts will decrease rapidlyhowever, when the plates are kept aerobically for

some time before incubation. In our laboratory, werecommend the use of peptone water with salineŽ .0.85% for the enumeration of bifidobacteria fromdairy product samples

It should be noted that the plating technique canmake a significant difference to the results of enu-meration of bifidobacteria. The pour-plate techniqueseems to be preferred to the spread-plate techniquefor enumeration of bifidobacteria in fermented dairyproducts. However, a study carried out by Payne et

Ž .al. 1999 to compare spread- and pour-plate tech-niques for the enumeration of three species of bifi-

Ždobacteria B. longum, B. adolescentis and B. bi-.fidum indicated that spread plates gave higher

recoveries than the pour plates. The differences inpercentage recovery observed using the spread- andpour-plate techniques are not easily explained but tosome extent reflect the requirement of these organ-isms for an anaerobic environment. It would there-fore be expected that higher recoveries would beachieved using the pour-plate technique. However,sensitivity to oxygen is different among different

Ž .strains and species Scardovi, 1986 . It is thus impor-tant to compare spread- and pour-plate techniques to

Table 1Survey on various diluents used for the enumeration of bifidobacteria in fermented dairy products

Diluent References

Ž .Peptone 0.1% water Arroyo et al., 1995; Collins and Hall, 1984; Ghoddussi andRobinson, 1996; Ingham, 1999; Kneifel et al., 1993;Laroia and Martin, 1991; Payne et al., 1999; Shah et al.,1995; Shin et al., 2000

Ž .Peptone 0.15% water Dave and Shah, 1996ŽPeptone–saline solution, 0.1% peptone Blanchette et al., 1996; Collins and Hall, 1984; Gomes

.and 0.9% NaCl et al., 1995Physiological saline solution Collins and Hall, 1984; Iwana et al., 1993

Ž .Tryptone 0.1% water Rybka and Kailasapathy, 1995ŽTryptone–salt, 0.1% tryptone and 0.9% Lapierre et al., 1992

.NaClŽ . .Tween 80 0.1% , peptone 1% Chapon and Kiss, 1991

phosphate buffer solutionŽ1.122% Na HPO , 1.122%,2 4

.0.05% KH PO2 4

Phosphate buffer solution Wijsman et al., 1989Ž0.6% Na HPO4, 0.45% KH PO ,2 2 4

.0.01% agar, 0.05% L-cysteine HClŽ .Phosphate buffer solution, Collins and Hall 1984 ; Ingham, 1999

Ž .0.06% Na HPO 0.03% KH PO2 4, 2 4Ž .Ringer Solution quarter-strength Asperger and Saad, 2000Ž .Ringer Solution quarter-strength Nebra and Blanch, 1999

q0.05% L-cysteine HCl


select the combination of medium and plating tech-nique that gives the most accurate representation of

Ž .the bifidobacteria viable count Payne et al., 1999 .

3. Non-selective culture media

Non-selective media are useful for routine enu-meration of bifidobacteria when present in non-fer-mented milks and in fermented milks containingbifidobacteria in order to determine the initial inocu-lum and to ascertain the length of time these organ-

Žisms remain viable during storage Rasic and Kur-mann, 1983; Samona and Robinson, 1991; Arroyo et

. Ž .al., 1994 . Scardovi 1986 indicated that, as bifi-dobacteria vary widely in their physiological require-ments for growth, no one selective medium is appro-

Ž .priate for all species Modier et al., 1990 . Thepreference should be given to substrates that permitsatisfactory growth of the largest number of bifi-dobacteria types presently known. The ingredients ofchoice are trypticase and phytone according to Scar-

Ž .dovi 1986 who suggested the use of a complex,Žnon-selective medium Trypticase-Phytone-Yeast ex-

.tract or TPY , which will allow the growth of allBifidobacterium species as well as other lactic acid

Ž .bacteria. Beerens 1990 observed that liquid enrich-ment media TPY and BHI exhibited identical results.

Ž .Roy and Ward 1992 indicated that TPY and MRS

may be used as enrichment media to support theŽ .growth of bifidobacteria Table 2 .

Table 2 shows basal culture media recommendedfor the cultivation of bifidobacteria. Bacterial num-ber in synthetic media or low-pH media can be lowerthan in the broths with a complex composition and in

Ž . Žthose with higher pH values 6.8–7.4 Pacher and. Ž .Kneifel, 1996 . The liver-cysteine-lactose LCL agar

Ž .of Blaurock was chosen by Lapierre et al. 1992 asa basis because it contains the most important ele-ments for bifidobacteria: lactose, liver infusion, pep-tone. However, the preparation of a liver infusion for

Ž . Ž .Blood–Liver BL broth and Blaurock LCLmedium is a messy task and can, therefore, not berecommended for routine work. The modified MRSŽ .MRS containing cysteine-HCl medium can provideoptimal overall growth conditions for the bifidobac-teria. L-cysteine is added to lower the oxidorreduc-

Ž .tion OrR potential in culture media and providesbetter anaerobic conditions for the growth of bifi-dobacteria. This amino acid is also regarded as anessential nitrogen source for bifidobacteria.

The solid non-selective media commonly used fordetection of bifidobacteria are: reinforced clostridial

Ž .medium agar RCM and RCM plus lactose andŽ .sheep blood mRCM ; BL agar, BL agar without

Ž .blood, Tryptone phytone yeast extract agar TPY ,Ž .modified MRS agar Table 2 . One of the first useful

media for the enumeration of bifidobacteria was

Table 2Basal and elective culture media used for bifidobacteria

Medium Medium name Final concentrations of Comments Referencesabbreviation additives

MRS De Man Rogosa Sharpe None Basal De Man et al., 1960TPY Tryptone Phytone Yeast None Basal Scardovi, 1986BL Glucose Blood–Liver None Basal Mitsuoka et al., 1965CLB Columbia None Basal Ellner et al., 1958

Ž .LCL Liver Cystine Lactose Blaurock None Basal Rasic and Sad, 1990RCM Reinforced Clostridia Medium None Basal Hirsch and Grinsted, 1954mMRS Modified MRS L-cysteine HCL, 0.05% elective Sykes and Skinner, 1973mMRSqblood Modified MRS L-cysteine HCL, 0.05% elective Pacher and Kneifel, 1996

Sheep blood 10 mlaX-a-Gal MRS X-a-Gal elective Chevalier et al., 1991

mBL Modified BL without blood L-cysteine HCL, 0.05% elective Arroyo et al., 1994mRCM Modified RCM Lactose 1.0% Elective Reuter, 1990

Human blood 50 ml differentialRCPB RCM Prussian Blue 0.03% differential Onggo and Fleet, 1993

a X-a-Gals5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside.


Lactobacillus agar modified from the medium ofŽ .Reuter by Klupsch Wijsman et al., 1989 . Reuter

Ž .1990 proposed Reinforced Clostridia MediumŽ .RCM plus 1% lactose supplemented with 5% bloodor 2% erythrocytes concentrate for total anaerobiccounts and for the separation of bifidobacteria inyogurt-like products.

Mitsuoka suggested the use of BL agar for non-selective enumeration of bifidobacteria from dairyproducts and intestinal materials as cited by Modier

Ž .et al. 1990 . This procedure was described in moreŽ .detail by Laroia and Martin 1991 ; however, the

described formulation is incomplete as the defibri-Žnated blood component 50 ml of horse or sheep

y1 .blood l of cooled medium was inadvertentlyomitted from the original formulation of Teraguchi

Ž .et al. 1978 .ŽAlthough modified MRS agar MRSq0.5% L-

.cysteine HCl is not selective for bifidobacteria, thismedium is recommended for their enumeration from

Ž .pure cultures. Modified BL mBL agar is also usedbecause this medium has been proven in severallaboratories to provide accurate counting of bifi-

Ž .dobacteria. Dave and Shah 1996 indicated thatMRS agar provided an excellent recovery of bifi-dobacteria. Commercially available media, mMRSand RCM and laboratory prepared medium, mBLagar, supported excellent growth of the strains ofbifidobacteria; however, this latter medium is not astime or cost effective as mMRS and RCM agarsŽ .Arroyo et al., 1994 . The percentage recovery ob-

Ž .tained by Payne et al. 1999 on mMRS for B.longum was 59% by spread plate and 45% by pourplate; for B. adolescentis, it was 92% by spread plateand 45% by pour plate and for B. bifidum, it was117% by spread plate and 107% by pour plate.

Ž .Therefore, Payne et al. 1999 concluded that mMRSmight be useful for enumeration of pure cultures ofbifidobacteria, particularly B. bifidum. Although Re-

Ž .inforced Clostridial Agar RCM gave higher recov-eries for B. longum and B. adolescentis. According

Ž .to Dave and Shah 1996 , recovery of all the bifi-dobacteria obtained on RCM agar was almost identi-cal to that on mMRS agar at pH 6.8, but the recoverywas slightly lower on RCM agar at pH 5.3. Hence,as they are commercially available RCM and mMRSwould likely be the media of choice for industrialquality control laboratories.

4. Improvement of elective properties

Bifidobacteria may be stimulated by short-chainfatty acids, like propionate and butyrate. BeerensŽ . Ž .1990 and Pacher and Kneifel 1996 modified thecomposition of culture media in order to improve the

Ž .growth conditions for bifidobacteria. Beerens 1990noted that the addition of propionic acid to Columbiaagar at pH 5.0 enhanced the growth of bifidobacte-

Ž .ria. Hartemink et al. 1996 found that low propi-Ž y1 .onate concentrations 10 and 15 g l enhanced the

growth of Lb. acidophilus whereas an increase ofpropionate concentration to 20 or 25 g ly1 inhibitedonly the Lb. acidophilus strains from dairy products.With 30 g ly1 of propionate, some strains of bifi-dobacteria were inhibited. Addition of butyrate didnot increase the selectivity of medium whereas theaddition of valerate resulted in inhibition of two outof six strains of bifidobacteria.

Certain substrates components such as yeast ex-tract, peptone, cysteine and starch are essential forthese microorganisms and were incorporated in

Ž .mMRS Pacher and Kneifel, 1996 . Moreover, yeastextract was considered an excellent growth promoter

Ž .by Ibrahim and Bezkorovainy 1994 . According toŽ .Pacher and Kneifel 1996 , the supplementation of

Žmodified MRS with water-soluble vitamins thiamin,.riboflavin and pantothenic acid and lactulose syrup

in addition to glucose, improved the growth of cer-tain Bifidobacterium species. Several growth pro-moting substances derived from human and bovinewhey might also be added to culture media becauseboth a-lactalbumin and b-lactoglobulin are good

Žbifidobacterial promoters Ibrahim and Bezko-.rovainy, 1994 , Indeed, the supplementation of the

Ž .modified MRS medium of Pacher and Kneifel 1996with human whey was very effective on growth ofbifidobacteria.

The exploitation of certain enzymatic propertiesof bifidobacteria might offer a promising tool fortheir specific detection via chromogenic reactions of

Žthe bacterial colonies Chevalier et al., 1991; Pacher. Ž .and Kneifel, 1996 Table 2 . The presence of a-

galactosidase could be a useful and simple methodfor rapid identification of bifidobacteria. Chevalier et

Ž .al. 1991 developed a method using a chromogenicsubstrate for the differential enumeration of bifi-dobacteria and lactic acid bacteria. Blue colonies are


seen on solid media due to the presence of a-galactosidase activity because the enzyme actionsplits the chromogenic substrate 5-bromo-4-chloro-

Ž .3-indolyl-a-D-galactoside X-a-Gal and released in-dol that imparts a blue colour to bifidobacterialcolonies producing a-galactosidase. However, Pacher

Ž .and Kneifel 1996 concluded that this reaction mightbe applicable only to a limited number of bifidobac-teria with relatively high a-galactosidase activity. Inaddition, Lb. acidophilus and lactococci may alsoproduce a-galactosidase thus leading to misinterpre-tation.

Oligosaccharides are used as bifidogenic factorsto stimulate the growth of bifidobacteria. It is as-sumed that these carbohydrates are selectively fer-mented by bifidobacteria. Oligosaccharides with bifi-dogenic activity are: Lactulose, trans-galactosyl

Ž .oligosaccharides TOS , fructo-oligosaccharidesŽ .FOS , isomalto-oligosaccharides, raffinose and soy-bean oligosaccharides. a-Galactosaccharides such asmelibiose and raffinose are metabolized by bifi-dobacteria and not by lactococci. a-Galactosidaseactivity of bifidobacteria participates in the first

Ž .degradation of these saccharides Sakai et al., 1987 .Raffinose is available in large quantifies and high

Ž .purity. Roy et al. 1997 proposed modified CAB-raffinose-based media for the enumeration of bifi-dobacteria in fresh cheese. The same principle has

Ž .been used by Hartemink et al. 1996 . Replacingraffinose by other carbohydrates such as lactulose,

Ž .fructo-oligosaccharides FOS and TOS did not re-sult in increased selectivity. TOS showed similarresults as raffinose, but with increased growth of B.bifidum. However, TOS is as yet not available com-mercially in large quantifies and high purity.

5. Differential culture media

Several media have been developed for differen-tial enumeration of bifidobacteria from other lactic

Ž .acid bacteria. Charteris et al. 1997 also revieweddifferential plating methodologies used for isolation,identification and enumeration of potentially probi-otic lactobacilli and bifidobacteria. Tamime et al.Ž .1995 noted that acidified media such as MRS orRogosa agar have been used successfully by someresearch groups for enumeration of bifidobacteria in

mixed cultures while others have reported their lim-ited selectivity. Reinforced Clostridial Prussian Blue

Ž .Agar RCPB was recommended by Ghoddussi andŽ .Robinson 1996 for the differential enumeration of

B. delbrueckii subsp. bulgaricus, Streptococcus ther-mophilus and bifidobacteria. B. bifidum and B. ado-lescentis appeared as white colonies while Lb. del-brueckii subsp. bulgaricus and S. thermophilusformed pale blue colonies surrounded by wide royalblue or thin light blue zones, respectively. Higherpercentage recoveries of bifidobacteria were achievedby pour plate than by spread plate on this mediumŽ .Payne et al., 1999 . Although the appearance of thecolonies was as described by Ghoddussi and Robin-

Ž .son 1996 when pure cultures were plated onto theŽ .medium, Payne et al. 1999 noted that it was ex-

tremely difficult to distinguish one species fromanother when mixed cultures were used.

Tryptose Proteose Peptone Yeast extract qŽ .Prussian Blue Agar TPPYqPB is another differ-

ential agar that might allow Lb. delbrueckii subsp.bulgaricus, S. thermophilus, bifidobacteria and Lb.acidophilus to be enumerated on one medium. Ac-

Ž .cording to Ghoddussi and Robinson 1996 , bifi-dobacteria gave white colonies, S. thermophilusformed pale blue colonies surrounded by a thin paleblue zone, Lb. delbrueckii subsp. bulgaricus pro-duced small white colonies surrounded by wide royalblue zones and Lb. acidophilus produced large paleblue colonies surrounded by a wide royal blue zone.

6. Selective culture media

For the purpose of proper and correct enumera-tion, various attempts have been made to obtain aselective medium for isolating Bifidobacterium spp.from other dairy-related microorganisms, especially

Ž .streptococci and lactobacilli Sozzi et al., 1990 .Methods to detect and enumerate bifidobacteria inproducts containing a mixed bacterial population arebased on the use of more or less traditional media forlactic acid bacteria or anaerobes, supplemented withvarious selective agents or a single carbon sourcethat inhibit or reduce the growth of other lactic acid

Žbacteria Teraguchi et al., 1978; Resnick and Levin,1981; Munoa and Pares, 1988; Cole and Fuller,1989; Wijsman et al., 1989; Beerens, 1990; Lapierre


Table 3Selective media used for the enumeration of bifidobacteria in fermented dairy products

Media Base Selectivity based on, ReferencesaNPNL agar BL NPNL solution Teraguchi et al., 1978aMRS–NPNL MRS NPNL solution Dave and Shah, 1996

TOS–NPNL NrA neomycin, paromomycin, Wijsman et al., 1989nalidixic acid, LiCl

Ž .TPYqNPNL TPY neomycin, paromomycin, Ghoddussi and Robinson 1996nalidixic acid, LiCl

BL-OG BL Oxgall, Gentamycin Lim et al., 1995RMSqPPNL Rogosa agar Sodium propionate, Neomycin, Samona and Robinson, 1991

Paromomycin, LiClMRSqDic MRS Dicloxacillin Sozzi et al., 1990TPYqDic TPY Dicloxacillin Sozzi et al., 1990mCAB Modified Propionic acid, 5 ml Beerens, 1990, 1991

Columbia agar baseEcobion 2 CAB Neomycin, LiCl Chapon and Kiss, 1991DP CAB Dicloxacillin, Propionic acid, 5 ml Bonaparte, 1997

aLP LCL LP mixture Lapierre et al., 1992aRAF 5.1 CAB LP mixture Roy et al. 1997

RB LiCl, Sodium propionate Hartemink et al., 1996BFM NrA LiCl, Methylene blue, Propionic Nebra and Blanch, 1999

acid, 5 mlGL agar NrA LiCl Iwana et al., 1993BIM-25 RCM Nalidixic acid, Polymycin B, Munoa and Pares, 1988

Iodoacetic acid,2,3,5-triphenyltetrazolium chloride

AMC RCM Nalidixic acid, Polymycin B, Arroyo et al., 1994, 1995Iodoacetic acid,

a2,3,5-riphenyltetrazolium chloride, LP mixture

a y1 y1 Ž y1 .Made from stock solution as follows: LP mixturesLiCl, 2 g l ; sodium propionate, 3 g l . NPNL solutions l Neomycinsulphate, 100 mg; Paromomycin, 200 mg; Nalidixic acid, 15 mg; LiCl, 3 g.

et al., 1992; Kneifel et al., 1993; Arroyo et al., 1995;.Lim et al., 1995 . These media contain bifidogenic

sugars or antibiotics that may cause lower viablenumbers of bifidobacteria and usually contain lactoseor glucose as a carbohydrate substrate, which en-hances the growth of all the bacteria used for the

Ž .production of yogurts Iwana et al., 1993 . In manycases, the enumeration of low numbers of bifidobac-teria might still be subject to interference by S.thermophilus, yielding pinpoint colonies. Generally,the selective media for bifidobacteria control thegrowth of lactic acid bacteria at dilutions higher than10y5.

The methods to determine the accurate viablecounts of bifidobacteria in food products are impor-tant, but controversial; some selective agar mediawere developed to enumerate bifidobacteria in fer-mented or unfermented dairy products, which may

contain two or three strains of lactic acid bacteria,especially Streptococcus spp. and Lactobacillus spp.Most of them have complex compositions that in-clude antibiotics as growth inhibitors, require longincubation times, or show low bacterial recoverylevels. These factors hinder their routine use for theenumeration of bifidobacterial populations in dairy

Ž .product Nebra and Blanch, 1999 . Table 3 showsprincipal selective media used for enumeration ofbifidobacteria in dairy products.

7. Comparative studies

Ž .Scardovi 1981, 1986 has previously reviewedand discussed several complex media, and mediacontaining a wide variety of antibiotics to select forenumeration of Bifidobacterium species. In order to


improve selectivity, antibiotics and other ingredientshave been used: kanamycin; neomycin, paro-momycin, sodium propionate, lithium chloride; sor-bic acid and sodium azide. Few media are trulyselective for bifidobacteria and some of them are

Žtime-consuming to prepare Pacher and Kneifel,.1996; Lim et al., 1995 . Reliable enumeration of

bifidobacteria seems to be achieved only if a micro-biologist knows which particular Bifidobacterium

Žstrain was used in the product Pacher and Kneifel,.1996 .

7.1. NPNL-based media

Ž .Teraguchi et al. 1978 developed an improvedselective medium for the enumeration of bifidobacte-ria in dairy products, which contained mixtures ofbifidobacteria, lactobacilli and streptococci. Thismedium called NPNL was BL-agar with neomycinsulfate, paromomycin sulfate, nalidixic acid andlithium chloride and was then stated by Laroia and

Ž .Martin 1991 . NPNL agar is considered by manyauthors to be the reference medium for the isolationof bifidobacteria from fermented dairy productsŽModier et al., 1990; Lapierre et al., 1992; Lim et al.,

.1995; Tamime et al., 1995 because it seems thatthese bacteria grow well in this medium. Neomycinsulphate and nalidixic acid were included as selec-tive agents to inhibit growth of Gram-positive andGram-negative rods, respectively. Lithium chlorideis a substance commonly used for the selective isola-tion of bifidobacteria. Although lithium chloride iscommonly used in microbiology, its mechanism ofaction on bacterial cells is still poorly understood.

Ž .Teraguchi et al. 1978 suggested that LiCl at aconcentration of 3 g ly1 restricted the growth of Lb.acidophilus and had little effect on the growth of B.bifidum.

Some authors noted that NPNL agar showed aŽgood selectivity towards bifidobacteria Wijsman et

.al., 1989; Lapierre et al., 1992 . Recovery of bifi-dobacteria on NPNL agar with blood was around90% and recovery of lactic acid bacteria was lessthan 2% as compared with BL agar without blood

Ž .according to Rasic and Sad 1990 . However, Ghod-Ž .dussi and Robinson 1996 observed that although

bifidobacteria grew well on BL medium withoutselective agents, only B. bifidum gave acceptable

growth as compared to B. adolescentis on NPNLŽ .agar. However, Shah et al. 1995 noted that the

NPNL agar restricted the growth of B. infantis andŽB. adolescentis. Finally, although IDF Rasic and

. ŽSad, 1990 suggested the use of NPNL agar Tera-.guchi et al., 1978 for isolation of bifidobacteria

from fermented milks, the medium was not includedŽ .in the comparative study of Bonaparte 1997 for the

Ž .following three reasons. 1 Preliminary trials showedŽ .that the recovery rates productivity of some Bifi-

dobacterium species was to low. The undesirableinhibitory effect was also mentioned by various au-

Ž .thors Pacher and Kneifel, 1996; Lim et al., 1995 .Ž . Ž .2 According to Lim et al. 1995 , NPNL seems to

Ž .have weak inhibitory effects on S. thermophilus. 3Finally, it is time-consuming to prepare the NPNLagar, because it contains 24 ingredients, some ofwhich must be filter-sterilized. Moreover, Laroia and

Ž .Martin 1991 indicated that careful measurement ofingredients and treatment of the NPNL medium isextremely important and necessary for consistent andreliable results.

MRS–NPNL agar is widely used for the recoveryand enumeration of bifidobacteria by researchers and

Ž .quality control laboratories. Dave and Shah 1996found poor recovery of B. adolescentis and B. pseu-

Ž .dolongum 20099 strains in MRS–NPNL mediumŽ .DSM 20099 . Therefore, the counts obtained onMRS–NPNL agar may not be representative of theviable cells that are present in the product, whichsuggests a need to countercheck the efficacy of thismedium with pure cultures before the medium isadopted for enumeration purposes. Wijsman et al.Ž .1989 observed that the addition of the mixture offour antibiotics in the same concentration as used inNPNL agar to trans-galactosylated oligosaccharideŽ .TOS medium inhibited the colony formation ofbifidobacteria completely, whereas an addition of theantibiotic mixture at a relative concentration of 30%NPNL had no effect on the colony count of bifi-dobacteria but inhibited the growth of pinpointcolonies of the streptococci.

7.2. BL-OG

Many authors have developed selective mediabased on the use of commercial medium or with asimpler composition than NPNL agar. Lim et al.


Ž .1995 proposed the use of BL agar containing ox-Ž . Ž . Žgall 0.2 mgrml and gentamycin 30 mgrml BL-

.OG agar , which is much simpler to prepare thanNPNL agar and gives higher recovery of bifidobacte-ria in yogurts. Oxgall at 0–2 mg mly1 was used toinhibit other non-intestinal lactic acid bacteria. Gen-tamycin, that, in studies on antibiotic sensitivity of37 strains of bifidobacteria and 58 strains of lacticacid bacteria, was found to have optimal selectivity

Ž .for bifidobacteria Lim et al., 1995 . Recovery ofbifidobacteria on BL-OG agar was around 90% orhigher compared with that on BL agar. All testedstrains of Lb. acidophilus, Lb. delbrueckii subsp.bulgaricus and Lb. casei were inhibited in BL-OGand NPNL. Strains of streptococci were inhibited onBL-OG whereas the NPNL agar had fewer inhibitoryeffects on S. thermophilus strains.

Ž .Payne et al. 1999 obtained comparable with thepour-plate results. BL-OG also showed good inhibi-tion to yogurt culture and Lb. acidophilus. Lim et al.Ž .1995 stated that BL-OG is simpler to prepare thanNPNL and gives a higher recovery of bifidobacteria.

Ž .However, Payne et al. 1999 indicated that NPNLgave higher percentage recoveries than BL-OG forB. longum by spread and pour plate and of B.bifidum by spread plate. They concluded, as forNPNL, the major disadvantage of this medium is thatit is time-consuming to prepare.

7.3. RMS agar

Ž .Samona and Robinson 1991 found that modifiedŽ . ŽRogosa agar RMS agar with PPNL sodium propi-

onate, paromomycin sulphate, neomycin sulfate and.lithium chloride gave the highest counts of bifi-

dobacteria without any growth of lactobacilli andstreptococci as compared to TPY with NNLŽneomycin sulfate, nalidixic acid and lithium chlo-

. Ž .ride . However, Rasic and Sad 1990 observed thatthe numbers of bifidobacteria on RMS agar wereslightly less than on modified Rogosa’s non-selectiveŽ .pH 5.9 and BL agar. Ghoddussi and RobinsonŽ .1996 observed that RMS with PPNL was inhibitoryfor B. bifidum and B. animalis. The same patternwas repeated on TPY with NPNL and PPNL. Theuse of a lower concentration of NPNL gave goodrecovery for both bifidobacteria while at the sametime the growth of cheese and yogurt cultures was

inhibited. These authors concluded that the concen-tration of antibiotic should be carefully adjusted toenumerate the precise strains being employed for anygiven product.

7.4. Other antibiotic-based media

The antimicrobial activity of 30 antibiotics towardlactic acid bacteria was examined by Sozzi et al.Ž .1990 using MRS and TPY media. Only nafcillinand dicloxacillin were found to inhibit streptococci

Ž .and lactobacilli. Wijsman et al. 1989 found thatMRS-nafcillin agar showed no inhibition of strepto-

Ž .cocci and lactobacilli whereas Sozzi et al. 1990indicated that MRS and TPY media supplementedwith 2 mgrl dicloxacillin behave similarly to NPNLagar medium.

Ž .Cole and Fuller 1989 proposed the use of Bifi-Ž .dobacterium selective medium BBM , which is MRS

medium without the acetate and with the addition ofcysteine raffinose, fructose and galactose, nalidixicacid and rifampicin. However, this medium is nottotally selective for bifidobacteria and dilution counton this semi-selective medium followed by assay ofthe amount of acetic acid produced under standardconditions should be done for enumeration of bifi-dobacteria.

Ž .Pacher and Kneifel 1996 developed a culturemedium for the detection and enumeration of bifi-dobacteria in fermented milk products by using MRSwith cysteine-HCl, lactulose and whey from humanmilk plus a selective supplement of aztreonam,nalidixic acid, netilmicin and paromomycin sulfate.This medium reliably enumerated bifidobacteria.Sometimes, streptococci produced pinpoint colonies,which could be easily distinguished from large bifi-dobacterial colonies. According to these authors, theirmedium allowed the enumeration of a wider spec-trum of bifidobacteria than the other selective mediapreviously described.

Two media for selective enumeration of bifi-dobacteria were investigated by Rada and KocŽ .2000 : Wilkins–Chalgren agar with addition of

Ž . Ž .neomycin 100 mgrl , paromomycin 200 mgrl ,Ž . Žnalidixic acid 15 mgrl and lithium chloride 3 grl;

.NPNL agar and Wilkins–Chalgren agar withŽ .mupirocin 100 mgrl: Mupirocin agar . Mupirocin

susceptibility testing enabled these authors to differ-


entiate bifidobacteria from other lactic acid bacteriatested. While lactobacilli, lactococci, streptococci andleuconostocs were consistently susceptible, no bifi-dobacteria isolate produced a visible zone around thedisc containing 200 mg of mupirocin. Mupirocinagar was both more elective and more selective incomparison with NPNL medium. The total counts onmupirocin agar were practically identical with thosefound on the control Wilkins–Chalgren agar, whilethe NPNL medium gave significantly lower numbersof bifidobacteria. All of the fermented dairy productsexamined contained 105 to 107 cfu bifidobacteriarmland recovery on NPNL was significantly less than onMupirocin agar, except for one fermented milk prod-ucts. Mupirocin completely inhibited lactobacilli anddairy cocci, but not bifidobacteria.

7.5. Modified Columbia agar

Ž .Beerens 1991 demonstrated that when propionicŽ .acid was added to Colombia agar mCol at pH 5.0,

it enhanced the growth of bifidobacteria. This agarwas described by Beerens as selective and elective

Ž .for bifidobacteria. Chapon and Kiss 1991 indicatedthat CAB medium with lithium chloride and

Ž .neomycine sulfate Ecobion 2 was more selectiveŽ .that the medium proposed by Beerens 1990 . How-

ever, mCAB is more appropriate for the enumerationof a large number of strains of bifidobacteria. More-

Ž .over, Silvi et al. 1996 noted that mCol seems to bethe most appropriate for this purpose. mCol wasfound to be the most selective, inhibiting the growthof six out of nine non-bifidobacteria strains tested,while at the same time allowing enumeration of fourout of five Bifidobacterium strains tested. However,

Ž .Payne et al. 1999 found that there was poor recov-ery of B. longum and no growth of B. bifidum onthis medium.

Ž .Bonaparte 1997 proposed a new combinationcalled DP medium which was based on the use of

Ž .Columbia agar with propionic acid 5 ml and di-Ž .cloxacillin 2 mgrl as additives. The pH was ad-

justed to 6.8 with NaOH. This medium allows thegrowth of bifidobacteria with a good productivityrate whereas the growth of Lb. acidophilus, S. ther-mophilus and Lb. delbrueckii subsp . bulgaricus was

Ž .prevented. Roy et al. 1997 found that viable countsof bifidobacteria on modified Columbia agar base

Ž .CAB with 0.05% cysteine-HCl plus raffinoseŽ .0.5% containing various selective agents werecomparable to those on non-selective media such asmMRS. They observed that the growth of S. ther-mophilus, Lb. acidophilus and Lb. bulgaricus strainswas inhibited on the mCol plus raffinose medium.

7.6. Lithium chloride–sodium propionate-based agar

7.6.1. LP agarA combination of 2 g ly1 of lithium chloride and

3 g ly1 of sodium propionate was added by LapierreŽ . Ž .et al. 1992 to liver-cystine-lactose LCL agar to

suppress the growth of lactic acid bacteria and toallow the selective enumeration of bifidobacteriafrom commercial products. Sodium propionate isused as a selective agent for the isolation of bifi-dobacteria. Bifidobacteria were selectively isolatedfrom all dairy products tested with LP agar in num-ber similar to those obtained with NPNL agar usedas reference medium. LP agar was hence comparablewith NPNL agar in terms of the numbers of bifi-dobacteria enumerated from dairy products, but ithad the advantages of to be simpler to prepare and

Ž .requiring a shorter incubation Lapierre et al., 1992 .Bifidobacteria tested also showed similar growth onLP agar and on TJA used as non-selective medium,demonstrating that the concentration of lithium chlo-ride and sodium propionate chosen had no inhibitoryeffect. In contrast, all strains of lactobacilli werecompletely inhibited but some mesophilic strains oflactococci were resistant to the concentrations oflithium chloride and sodium propionate used.

Ž .Lapierre et al. 1992 proposed the incubation ofplates at 408C to prevent the growth of these strains.

In their study to compare the performance of sixselective media for the enumeration of three species

Ž .of bifidobacteria in pure culture, Payne et al. 1999should replace incubation of plates at 408C by anincubation at 378C due to the poor recoveries ob-served at the recommended incubation temperature

Ž .by Lapierre et al. 1992 . In addition, there wassignificant recovery of yogurt culture organisms.

Ž .Payne et al. 1999 concluded that LP proved to be apoor medium for selective enumeration of the strainsof B. longum and B. adolescentis used in their

Ž .study. Sanders et al. 1996 also evaluated LP agar inorder to enumerate each species of bifidobacteria


selectively from composite mixtures in milk. Bifi-dobacteria appeared to be somewhat more sensitiveto the selective agents; four of the five strains testedshowed statistically significant differences of 24 to77% reduction in counts between LP agar and thenon-selective medium reinforced clostridia agarŽ .RCM . In products formulated with frozen concen-trates cultures, freeze injury of the cells could resultin even more dramatic differences in counts betweenselective and non-selective media. Colony formationof psychrotrophs was observed on LP agar at 48Cand 108C at concentration that were significantlylower than plate count agar. They concluded that themedium is not ideally selective but should be satis-factory when used to enumerate lactic cultures dif-ferentially in products within 1 week of manufactureor in products that have not been stored at 108C.

7.6.2. RAF 5.1 mediumŽ .Five species of bifidobacteria 15 strains , two

strains of Lactococcus lactis ssp. lactis, two strainsof L. lactis ssp. cremoris, and one strain of L. lactisssp. lactis var diacetylactis were used by Roy et al.Ž .1997 to develop a selective medium for bifidobac-

Žteria. mCol plus raffinose with LP mixtures lithiumy1 y1.chloride, 2 g l and sodium propionate, 3 g l

with pH adjusted to 5.1 was used successfully as aŽ .selective medium RAF 5.1 for the enumeration of

bifidobacteria from fresh cheeses. Viable counts ofbifidobacteria on RAF 5.1 were comparable to thoseon mCol plus LP mixture. Hence, viability of bifi-

Ž .dobacteria on mCol Beerens, 1990 plus raffinoseŽagars containing various selective agents LPsRAF

5.1; gentamycinsRAF G; and NPNLsRAF-.NPNL were compared with that on non-selective

Ž y1 .mBL agar. The gentamycin 30 mg ml or NPNLŽ y1solution l : Neomycin sulfate, 100 mg; Paro-

.momycin, 200 mg; Nalidixic acid, 15 mg; LiCl, 3 gwas incorporated to mCAB plus raffinose to replacethe LP solution. The average colony counts of bifi-dobacteria on these modified raffinose-based selec-tive media were comparable to those on the BLmedium. Hence, RAF 5.1, RAF-G and RAF-NPNLcould inhibit lactococci, which can be used for themanufacture of fresh cheeses because raffinose is not

Ž .metabolized by lactococci. Roy et al. 1997 recom-mended RAF 5.1 because this medium is easier to

prepare than RAF with gentamycin or NPNL solu-tion. However, the use of mCAB plus lactose andgentamycin must be used for the selective enumera-tion of B. bifidum because some strains of thisspecies are raffinose negative.

7.6.3. RB mediumA new selective medium for the detection of

bifidobacteria was developed by Hartemink et al.Ž .1996 . The medium owes its selectivity to the pres-

Ž y1 . Žence of propionate 15 g l and lithium chloride 3y1 . Ž y1 .g l as inhibitory agents, and raffinose 7.5 g l

Žas a selective carbon source. In addition. casein 5 gy1 .l is used as a protein source, which results in a

zone of precipitation around the colonies of bifi-dobacteria. The medium, Raffinose-BifidobacteriumŽ .RB agar, is free of antibiotics and easy to prepare.Bifidobacteria growing on RB agar show a yellowcolony with a yellow halo and a precipitation zonearound the colony. All dairy bifidobacteria grew wellon RB agar, except for some B. bifidum strains. Nonon-bifidobacterial strains used in dairy productsgrow with the distinctive characteristics. Comparisonwith other media indicated that RB agar is moreselective than the other media used. The followingmedia for bifidobacteria were tested by Hartemink

Ž .and Rombouts 1999 for the determination of bifi-dobacteria in feces of human, cat and pig: mColŽ . Ž .Beerens, 1990 , RB medium Hartemink et al., 1996

Ž .and NPNL agar Teraguchi et al., 1978 . They con-cluded that none of the three media tested was veryselective.

7.6.4. BFMAnother selective antibiotic-free medium for Bifi-

dobacterium spp. was defined by Nebra and BlanchŽ .1999 . Lactulose is the main carbon source of Bifi-

Ž .dobacterium medium BFM , which includes methy-lene blue, propionic acid, and lithium chloride asinhibitors of some related bacterial species. In orderto measure the recovery on BFM agar, two mediawere used for comparison: Columbia Blood agarŽ .CBA supplemented with glucose and L-cysteine, anenriched medium, and a previously described Bifi-dobacterium medium, BL medium. The selectivityof BFM did not affect the growth of 23 of the 26Bifidobacterium strains tested. For most of the 26


Bifidobacterium strains, similar colony counts wereobtained on BFM, CBA and BL medium. Nebra and

Ž .Blanch 1999 concluded that the simple composi-tion of BFM makes it a feasible medium for routinemonitoring purposes. Its selectivity could provide adifferential tool for the recovery of bifidobacteria infood microbiology. Finally, the recovery on BFMagar suggests the practical use of BFM for enumera-tion of Bifidobacterium in routine monitoring offermented dairy products such as yogurts.

7.6.5. Galactose agarŽ . Ž .Iwana et al. 1993 prepared Galactose agar GL

for isolation and identification of Bifidobacteriumspp. in commercial yogurts sold in Europe. BL agarand GL agar were compared for their ability torecover Bifidobacterium spp. from pure cultures. Allthe Bifidobacterium strains tested grew well on bothmedia, and the counts on GL agar were almost thesame as those on BL agar. No strains of the faculta-tive aerobes, except for Lb. helÕeticus and Lb. caseiwere detected on GL agar. The results showed thatGL agar can be used for the selective enumeration ofbifidobacteria with addition of lithium chloride inlower concentrations than that of BS. The BS agarwas prepared by adding LiCl, paromomycin sulfate,neomycin sulfate and sodium propionate to BL agarŽ .Saito et al., 1992 .

7.7. BIM-25

Ž .Munoa and Pares 1988 developed a selectivemedium called BIM-25 for isolation and enumera-tion of Bifidobacterium spp. from water samplesbased on RCA supplemented with nalidixic acid,polymixin B sulfate, kanamycin sulfate, iodoaceticacid and 2,3,5-triphenyltetrazolium chloride. Iodoac-etate inhibits glyceraldehyde-3-phosphate dehydro-genase and reduces the growth of nonbifidobacterial

Ž .contaminant colonies. Silvi et al. 1996 observedthat BIM-25 was very selective, inhibiting six of thenine non-bifidobacteria strains tested, but it alsoinhibited the growth of Bifidobacterium strains. Ing-

Ž .ham 1999 compared modified BifidobacteriumŽ .iodoacetate medium mBIM , a modification of the

Ž .medium described by Munoa and Pares 11 to MRSagar with added cysteine, bile, and dicloxacillin

Ž .MRSqBCD , which is used commercially for enu-meration of Bifidobacterium spp.

7.8. AMC

Ž .Selective media evaluated by Arroyo et al. 1995Ž .included lithium chloride–sodium propionate LP

agar, modifications of BIM-25 agar and a compositeŽ .medium AMC agar . The BIM 25 was modified by

either reducing the iodoacetate concentration by oneŽ .half mBIM-1 or by completely omitting iodoac-Ž .etate mBIM-2 . The Arroyo, Martin and CottonŽ .Agar AMC agar was developed by adding the

selective components in LP agar to mBIM-1 agarŽ .Arroyo et al., 1995 . Lb. acidophilus was not inhib-ited by mBIM-1 whereas LP agar completely inhib-ited growth of Lb. acidophilus. AMC is a selectivemedium that supported the growth of B. longumŽ .Payne et al., 1999 while inhibiting the growth ofLactococcus lactis subsp. lactis, Lb. delbrueckiisubsp. bulgaricus and S. thermophilus. It was, how-ever, found to allow slight growth of Lb. acidophilusŽ .Arroyo et al. 1995 .

Ž .Arroyo et al. 1995 indicated that no viable L.lactis ssp. lactis were detectable using AMC agar,indicating that this medium completely inhibits L.lactis ssp. lactis. LP failed to inhibit L. lactis ssp.lactis because no significant differences were evi-

Ž .dent between the reference medium Elliker and LPagar. The LP agar is not satisfactory for inhibition of

Ž .S. thermophilus Payne et al., 1999 .Ž .Sanders et al. 1996 tested AMC agar on five

strains of bifidobacteria. All strains showed statisti-cally significant reductions in counts compared with

Ž .counts of the non-selective medium RCA althoughtwo strains seemed most dramatically inhibited. Thisobservation suggests that the AMC medium is not anoptimal medium for differential enumeration. Sanders

Ž .et al. 1996 concluded that additional research inthis area is required because several bifidobacteriashowed significant differences between the selectiveand non-selective media. On the other hand, on thebasis of performance as a selective medium for theenumeration of bifidobacteria, and ease of prepara-

Ž .tion, Payne et al. 1999 concluded that AMC provedto be the best choice for the routine enumeration ofbifidobacteria in mixed culture in dairy products.


8. Concluding remarks

The plating technique methodologies used for theenumeration of bifidobacteria can make a significantdifference to the results. The use of peptone water

Ž .with saline 0.85% can be recommended as diluentsolution for the enumeration of bifidobacteria fromdairy product samples. The pour-plate techniqueseems to be preferred to the spread-plate techniquefor enumeration of bifidobacteria in fermented dairyproducts; however, it is important to compare spread-and pour-plate techniques in order to select the com-bination of medium and plating technique giving themost accurate representation of the viable count ofbifidobacteria.

Commercially available reinforced clostridial agarŽ . Ž .RCA and mMRS MRS containing cysteine-HClmedia can provide optimal overall growth conditionsfor the non-selective enumeration of bifidobacteria.L-cysteine is added to lower the oxido-reductionŽ .OrR potential in culture media and provides betteranaerobic conditions for the growth of bifidobacteria.Oligosaccharides are used as bifidogenic factors tostimulate the growth of bifidobacteria. Raffinose thatis available in large quantifies and high purity can beadded to non-selective and selective media to im-prove their elective properties.

ŽThe medium called NPNL was BL glucose.Blood–Liver -agar with neomycin sulfate, paro-

momycin sulfate, nalidixic acid and lithium chlorideis considered by many authors to be the referencemedium for the isolation of bifidobacteria from fer-mented dairy products. However, Columbia agar basemedia with lithium chloride and sodium propionateplus raffinose or Columbia agar with propionic acidŽ . Ž .5 ml and dicloxacillin 2 mgrl as additives andMRS medium supplemented with neomycin, paro-momycin, nalidixic acid and lithium chloride can berecommended for selective enumeration of bifi-dobacteria in dairy products.

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media for the isolation and enumeration of bifidobacteria in dairy products

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