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148 A. WEISS et al.: Selective Media for Enumeration of Probiotic Enterococci, Food Technol. Biotechnol. 43 (2) 147–155 (2005)gram, provided that it is measurable by an official orscientifically valid method (3). Due to the importance ofenterococci in different foods, feeds, clinical and environmentalsamples, numerous media have been used forthe detection, isolation and enumeration (4). This studywas performed within an EU project initiated by a DedicatedCall and was based on the regulations of theCouncil Directive 70/524/EEC (2) concerning additivesin feeds and the application of probiotics in feeds. Accordingto the Official Journal of the European CommunitiesC 263/3 (5), eleven genera of bacteria, three generaof yeast and one mould have been approved fortemporary use within specified member states and arein line with the provisions of Council Directive 93/113/EC (3).Twelve media were selected based on a literaturesearch (4) and tested with a set of strains. The specieswere selected based on their involvement in probioticanimal feeds as outlined in the Official Journal of theEuropean Communities C 263/3 (5).Material and MethodsTest strainsThe strain set used for the selectivity test consistedof twenty-eight enterococci, eleven lactobacilli, six pediococciand one Streptococcus thermophilus strain (seeTable 1). Lactobacilli, pediococci and S. thermophilus werechosen as they are authorised by the EU to be incorporatedin feeds, and are so closely related to enterococcithat they might interfere with their enumeration.Culturing of test strainsThe test strains used were subcultured as follows:enterococci were grown in 10 mL of brain heart infusionbroth (BHI, CM 225, Oxoid) under aerobic conditionsfor 24 h at 37 °C. Lactobacilli were grown in MRS broth(1.10661, Merck) under anaerobic conditions (anaerobicchamber; atmosphere: N 2 80 %, CO 2 10 % and H 2 10 %)for 24 h at 37 °C. Trypticase soy broth (TS, CM 129, Oxoid)supplemented with 3 g/L of yeast extract (1.037753,Merck) was used for subculturing of the S. thermophilusstrain under anaerobic conditions at 32 °C for 24 h andfor pediococci under the same conditions for 48 h.Selective mediaThe tested selective media were prepared accordingto the author’s or manufacturers’ guidelines. After sterilisationthe media were cooled to 50 °C and plates wereprepared by pouring 17 mL of sterile medium into a sterilePetri dish. All media used are described below, includingimportant details of their preparation.Bile esculin (BE) agar (CM 888, Oxoid; 44.5 g/L),which was autoclaved for 15 min at 121 °C, was applied.Additionally, two bile esculin azide (BEA) agarsfrom different manufacturers were tested: enterococcosel(ECSA) agar (4312205, Becton-Dickinson; 56 g/L,which was autoclaved for 15 min at 121 °C) and D-coccosel(D-Cocc) agar (51025, bioMérieux; 56 g/L, whichwas autoclaved for 15 min at 121 °C).Table 1. List of test strainsStrain Code Genus – species – subspecies Other codeEn1 Enterococcus faecalis DSM 2570En2 E. faecalis DSM 20478En3 E. faecium DSM 2918En4 E. faecium DSM 20477En5 E. durans DSM 20633En6 E. gallinarum DSM 20628En7 E. faecium Own isolateEn8 E. faecium Own isolateEn9 E. faecium Own isolateEn10 E. faecium Own isolateEn11 E. faecium DSM 3320En12 E. faecalis DSM 2981En12A E. faecalis ATCC 14506En13 E. durans ATCC 6056En14 E. faecium ATCC 27270En15 E. faecalis ATCC 27274En16 E. faecium DSM 2146En17 E. faecium DSM 6177En18 E. avium DSM 20679En19 E. casseliflavus DSM 20680En20 E. hirae DSM 20160En21 E. malodoratus DSM 20681En22 E. mundtii DSM 4838En23 E. raffinosus DSM 5633En24 E. faecium DSM 7134En25 E. faecium NCIMB 10415En26 E. faecium NCIMB 11181En27 E. faecium NCIMB 30096En28 E. faecium Own isolateEn30 E. faecium ATCC 19434Lb1 Lactobacillus acidophilus DSM 20079Lb7 L. amylovorus DSM 20531Lb12 L. rhamnosus DSM 20021Lb13 L. reuteri DSM 20016Lb14 L. plantarum Own isolateLb17 L. fermentum ATCC 9338Lb20 L. casei Own isolateLb21 L. reuteri Own isolateLb22 L. casei DSM 20011Lb23 L. plantarum DSM 20174Lb28 L. rhamnosus DSM 7133Sc1 Streptococcus thermophilus DSM 20617Pd1 Pediococcus acidilactici DSM 20248Pd2 P. acidilactici DSM 20238Pd3 P. damnosus DSM 20331Pd4 P. parvulus DSM 20332Pd5 P. dextrinicus DSM 20335Pd6 P. pentosaceus DSM 20336Pd7 P. pentosaceus ATCC 43200Pd8 P. inopinatus DSM 20285
A. WEISS et al.: Selective Media for Enumeration of Probiotic Enterococci, Food Technol. Biotechnol. 43 (2) 147–155 (2005)149Cephalexin aztreonam arabinose (CAA) agar (accordingto Ford et al. (6)) consists of Columbia agar base40 g/L, arabinose 10 g/L and phenol red solution 3.6mL/L, m/V=2. The pH value was adjusted to pH=7.8and the medium was autoclaved for 20 min at 114 °C(0.7 bar). After the medium had reached 50 °C, freshsterile solutions of aztreonam and cephalexin were addedto reach the final concentrations of 75 and 50 mg/L,respectively.Chromocult enterococci (CE) agar (1.10294, Merck;Chromocult enterococci broth 18 g/L and agar 14 g/L)was autoclaved for 15 min at 121 °C.Citrate azide Tween carbonate (CATC) agar (1.10279,Merck; 56 g/L) was autoclaved for 15 min at 121 °C.When the medium had reached approximately 50 °C,filter-sterilised solutions were added: 20 mL of sodium--hydrogen carbonate m/V=10, 10 mL of 2,3,5-triphenyltetrazolium chloride (TTC) solution m/V=1 and 4 mL ofsodium azide solution m/V=10.mE (mE) agar (0333-17, Difco; 72.1 g/L) was autoclavedat 121 °C for 15 min. When the medium hadreached a temperature of approximately 50 °C, filter--sterilised solutions were added: 15 mL of TTC m/V=1and 0.24 g of nalidixic acid.Fluorescent gentamicin thallous carbonate (fGTC)agar (according to Littel and Hartman (7)) was obtainedby suspending CASO agar 40 g, potassium-dihydrogenphosphate5 g, amylose azure 3 g, galactose 1 g, thalliumazure 0.5 g, 4-methyl umbelliferyl-a-D-galactoside100 mg and 0.75 mL of Tween ® 80in1Lofdistilled waterand mixing thoroughly. Then 2.5 mL of gentamicinsolution (1 mg/mL) were added and autoclaved for 15min at 121 °C. When the medium had reached a temperatureof approximately 55 °C, 20 mL of filter-sterilisedsodium hydrogen carbonate solution (m/V=10) wereadded.Kanamycin esculin azide (KEA) agar (CM 591, Oxoid;42.6 g/L) was obtained by adding two vials of kanamycinsupplement (SR92, Oxoid), and then autoclaved for15 min at 121 °C.KF-streptococcus (KF) agar (CM 701, Oxoid; 76.4g/L) was autoclaved at 121 °C for 10 min. When themedium had reached 50 °C, 10 mL of filter-sterilisedTTC solution (m/V=1) were added.Membrane-filter enterococcus selective (SB) agar(according to Slanetz and Bartley (8); 1.05289, Merck;41.5 g/L) was sterilised by heating in a current of steamin an autoclave without excess pressure for 20 min.When the medium had cooled to approximately 50 °C,10 mL of filter-sterilised TTC (m/V=1) were added.Merckoplate Barnes (BA) agar (13576, Merck; ready--to-use plates) was used.Oxolonic acid esculin azide (OAA) agar (accordingto Audicana et al. (9)) was obtained by suspending 42.6g of kanamycin esculin azide agar (CM 591, Oxoid) and0.25 g of sodium azide in 1 L and then autoclaved for 15min at 121 °C. Oxolonic acid (0.1 g) was dissolved in 5mL of 0.1 M NaOH and filter-sterilised. The aliquots ofthe solution (1 mL of each) were stored at –20 °C. Whenthe medium had reached a temperature of approximately45 °C, 0.5 mL of oxolonic acid solution wereadded under aseptic conditions.Sterile media in Petri dishes, protected against dehydration,were stored for up to two weeks at a temperatureof 4 °C.Evaluation of selective mediaBased on a comprehensive literature review (4), thecited media were grouped according to their selectivecomponents. Already well-evaluated, mainly commerciallyavailable media were selected out of the maingroups to test their selectivity with a set of enterococciand strains of genera of other lactic acid bacteria. Thesubcultured strains were loop-streaked onto the agarplates. After incubation under aerobic conditions for 24h (BE, ECSA, D-Cocc, CAA, CATC, fGTC, KEA, SB, BA,OAA) or for 48 h (CE, mE, KF), the growth of the teststrains was evaluated. The colony size was measured(for parameters see Tables 2 and 3), then the growthwas described, and digital photographs of the wholeplates and close ups (microscope SZH10, Olympus) ofsingle colonies were taken (data not shown).Comparative testing of three selective mediaand BHI agarIn order to set up a protocol for the enumeration ofenterococci in feeds and feed supplements, the threemost selective media (ECSA, mE and CATC) were chosenand included in further trials. Their performancewas compared with the non-selective control mediumBHI concerning their productivity. The productivity wascalculated according to the following formula:N ssP =N Nsss0/1/number of colonies of the sought type obtained onselective mediumsN 0 number of colonies of the sought type obtained oncontrol mediumFrom a liquid culture of the strain E. faecium En24(contained in the feed supplement »Provita – LE«) oneloop was transferred into sterile BHI broth, blended andincubated under aerobic conditions for 24 h at 37 °C. Avolume of 1 mL of this turbid broth was used to preparea single series of decimal dilutions up to the dilution of1:10 7 . The dilutions of 1:10 5 , 1:10 6 and 1:10 7 were used toinoculate plates of the tested selective media and BHI(two parallel plates per dilution) using the surface platingmethod. The inverted plates were incubated underaerobic conditions for 24 h at 37 °C. The colony-formingunits (CFU) were determined right after the incubation.The arithmetic means of the colony counts of theplates with the optimum dilution (between 5 and 300colony-forming units per plate) and the productivitieswere calculated. The experiment was repeated with modifications(mE agar was no more included, the dilutionsof 1:10 6 , 1:10 7 and 1:10 8 were applied for inoculation,three plates were inoculated at the same time forthe dilution of 1:10 7 and two plates for each of the otherdilutions).
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