Comparison of Selective Media for the Enumeration of - Food ...

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152 A. WEISS et al.: Selective Media for Enumeration of Probiotic Enterococci, Food Technol. Biotechnol. 43 (2) 147–155 (2005)Bile esculin (BE) agarOn bile esculin (BE) agar, enterococci grew as coloniesof small to large size and white colour, only E. casseliflavusexhibited light brown and E. mundtii yellowpigmented colonies. The colonies of all strains were glistening.With the exception of E. avium, strong hydrolysisof esculin was observed. Seven strains of lactobacilli andsix strains of pediococci grew on BE medium. L. plantarumcould not be distinguished macroscopically fromenterococci, due to its ability to hydrolyse esculin andits small colony size.Bile esculin azide (ECSA) agarThis medium was one of the tested formulations ofbile esculin azide (BEA) agar. Most of the E. faeciumstrains grew on ECSA forming medium to large size colonies.The colonies of E. casseliflavus were brown andthe colonies of E. mundtii were yellow. All enterococciwith the exception of E. gallinarum exhibited strong hydrolysisof esculin. Three Lactobacillus and six Pediococcusstrains displayed minimal growth under the givenconditions. The strains L. plantarum, P. dextrinicus and P.pentosaceus, which showed strong hydrolysis of esculin,could be distinguished from enterococci by their colonysize.Bile esculin azide (D-Cocc) agarThis medium was the second of the tested formulationsof bile esculin azide (BEA) agar. Few enterococcishowed poor growth (small colonies), but most grew onD-coccosel agar as medium-sized colonies, exhibitingstrong hydrolysis of esculin. E. mundtii showed lightbrown colonies. Two strains of lactobacilli and fourstrains of pediococci were able to grow under the specifiedconditions, but did not hydrolyse esculin. Only L.plantarum exhibited strong esculin hydrolysis, and couldbe distinguished from enterococci by its colony size.Cephalexin aztreonam arabinose (CAA) agarOn CAA agar E. faecium grew as white colonies ofpinpoint to medium size. E. mundtii displayed yellowpigmented colonies. For some enterococci acidificationwas observed. Growth was detected for eight strains oflactobacilli and seven strains of pediococci. They allgrew as pinpoint colonies, some exhibiting acidification.Enterococci, therefore, could not be distinguished macroscopicallyfrom lactobacilli and pediococci on CAAmedium.Chromocult enterococci (CE) agarOn this agar all enterococci grew as blue colonies ofpinpoint to large size. Three strains of lactobacilli andfive strains of pediococci exhibited growth on this medium.L. casei, L. plantarum, P. dextrinicus and two strainsof P. pentosaceus could not be distinguished from enterococcimacroscopically.Citrate Tween carbonate azide (CATC) agarOn CATC medium, enterococci appeared as coloniesof various sizes (pinpoint to medium) and colours(rosy to dark red). Only enterococci were able to growon this medium, but on account of the small colonysizes the plates were difficult to evaluate. Under thegiven test conditions, CATC medium displayed the bestselectivity.mE (mE) agarEnterococci grew as pinpoint to large size coloniesof rosy to dark red colour on mE medium, some colonieswere thus too small for colony counting. The coloniesof the type strains of E. durans and E. gallinarumwere surrounded by light red halos. The type strain ofL. plantarum and five strains of pediococci grew on mEmedium. They were distinguishable from enterococci.Fluorescent gentamicin thallous carbonate (fGTC) agarMost enterococci grew on fGTC medium as whitecolonies. E. casseliflavus and E. mundtii exhibited yellowpigmented colonies. For some enterococci fluorescencewas observed. Seven strains of lactobacilli, six pediococcalstrains and S. thermophilus grew on fGTC medium.All these strains formed pinpoint-sized colonies andcould therefore be distinguished from enterococci.Kanamycin esculin azide (KEA) agarAll enterococci grew on KEA medium as white coloniesof pinpoint to large size except E. mundtii, whichexhibited yellow colony colour. For two strains of E. faecalisand for E. mundtii dark centres of the colonies wereobserved. With the exception of E. avium and E. malodoratus,all enterococci exhibited strong hydrolysis of esculin.Eight strains of lactobacilli and six strains of pediococcigrew on KEA medium. However, they could notbe distinguished from enterococci macroscopically.KF (KF) agarEnterococci grew as pinpoint to large size colonieson KF medium. The colour of the colonies ranged frompink to dark red. For the type strains of E. faecalis and E.hirae red and pink halos of the colonies, respectively,could be observed. Some strains of E. faecium formedcolonies with light zones. Acidification of the mediumsurrounding the colonies was observed by the change incolour of the indicator dye to yellow. Three strains oflactobacilli and five strains of pediococci grew on KFmedium, of which only P. dextrinicus could not be distinguishedmacroscopically from enterococci.Slanetz Bartley (SB) agarEnterococci grew on SB medium as rosy to dark redcolonies of pinpoint to medium size. Colonies of severalstrains of E. faecium were surrounded by light peripheries.Again, the enterococcal colonies were too small foreasy evaluation. Two strains of lactobacilli and five strainsof pediococci grew on SB medium. They all formed pinpoint-sized,white colonies. As the E. faecium strain En3showed these characteristics as well, these microorganismscould not be macroscopically distinguished fromenterococci.Merckoplate Barnes (BA) agarEnterococci grew on BA medium as colonies of pinpointto medium size. The colony colour ranged fromwhite via rosy to dark red. The colonies of several strainswere surrounded by light peripheries. Eleven strains oflactobacilli and seven strains of pediococci grew on BAmedium. They all exhibited white colony colour and
A. WEISS et al.: Selective Media for Enumeration of Probiotic Enterococci, Food Technol. Biotechnol. 43 (2) 147–155 (2005)153pinpoint or small colony size and could therefore not bedistinguished from enterococci.Oxolonic acid esculin azide (OAA) agarEnterococcal colonies grown on OAA medium wereof pinpoint to large size and of white colour, with theexception of E. mundtii, which produced yellow colonies.The colonies of the type strain of E. casseliflavus and ofE. faecium En24 showed dark centres. Three strains oflactobacilli and five strains of pediococci grew on OAA--medium. They could not be distinguished macroscopicallyfrom enterococci.Comparative testing of three selective media andBHI agarOn account of the results of this first test series, thethree selective media ECSA, CATC and mE were selectedfor further evaluation. The productivities of all threeselective media were close to 100 % (ECSA: 95.2 %,CATC: 111.9 % and mE: 96.4 %). Comparable resultswere obtained in the second test series (data not shown).Enterococci could be easily recognised on ECSA becausethey produced a dark brown halo surrounding the coloniesdue to the hydrolysis of esculin. Plates containingthis medium were easy to evaluate: most enterococciformed colonies of medium size (1 mm in diameter) underthe specified condition within 24 h. Plates of mEagar were difficult to read because of the very smallenterococcal colonies. Under routine laboratory conditionsthese plates would need to be incubated for atleast 48 h, thus this medium was abandoned. CATCplates showed very small colony sizes and lightly colouredcolonies as well. Because of the same reasons asfor the mE agar, this medium was excluded from furthertests.As this project aimed at developing a standard methodfor the routine analysis of enterococci in feeds, themain goal was to find a selective medium which is easyto prepare and yields productivity rates comparable tothe non-selective BHI medium. Furthermore, much importancewas attached to the selectivity and the electivityof the medium, minimising the effort of additionaltests for the differentiation and identification of potentialenterococci. Of the three selective media evaluatedin this test series, ECSA performed best on account ofthe colony sizes observed and of its good electivity. Thus,this medium was evaluated further in comparison withBHI medium.Comparative testing of BEA agar (ECSA) withBHI agarIn order to evaluate possible influences on the resultsof the used methods (surface-plating and pouring),and possible matrix influences on the results (e.g. feedpowder, feed flakes in comparison with pure cultures)further studies using ECSA and BHI were conducted.Apart from enterococci no other microorganisms wereobserved to grow on ECSA under the specified conditions,thus confirming the good selectivity of this medium.The relative coefficients of variation (CV) rangedfrom 2.62 to 13.41 %, only in three cases exceeding 10 %.This value was influenced by neither the sample nor themedium used nor the method (surface-plating or pouring).The means of the four different sets of values of eachtest (ECSA pouring and ECSA surface-plating method,BHI pouring and BHI surface-plating method) were enteredinto the LSD test. The calculated LSD values rangedbetween 1.32 and 3.35. For all sets of samples (exceptEn30, series 2) the null-hypothesis had to be dismissedat least once, indicating that the two values belong totwo sets of samples. This may indicate that differencesexist between the two media and the two methods.The productivities within a method and a seriesranged from 0.1 to 5.6 % for the surface-plating, andfrom 0.1 to 6.9 % for the pouring method. Higher productivityvalues were found for the pouring methodthan for the surface-plating method in three of the fourcases, though the differences between the results of thetwo methods were minute. This may be on account ofthe uniform exposure of the enterococci to oxygen. Forthis reason and because of its advantages in routine control,the surface-plating method was chosen for all furtherexaminations.Provisional standard for the enumeration ofenterococci in feedsBased on these findings a provisional standard protocol(bile esculin azide agar, surface-plating method,aerobic incubation for 24 h at (37±1) °C) was developed.For further method details see Leuschner et al. (11).DiscussionBased on an extensive literature review and on thefindings of Reuter and Klein (12), well-known mediawere selected and formed the basis of this investigation.For selectivity testing, sets of enterococci and other lacticacid bacteria (LAB) test strains were used. The LABstrains were selected in a way that their genus and speciescorresponded to the species that were also appliedas feed additives. The chosen twelve selective mediawere evaluated concerning their selectivity for enterococci.The test strains were loop-streaked onto the agarplates. After incubation for 24 or 48 h under aerobicconditions to enhance the selectivity, the growth of thestrains was evaluated.The agar evaluation (Tables 2 and 3) resulted in theselection of three agars for further testing. CATC displayedthe best selectivity, but several enterococcal teststrains were also inhibited in their growth (small colonies).These findings correspond to those of Ellerbroek(13), who compared CATC, BE and SB agars. ECSA andmE agar were chosen because of their good selectivityand the relatively pronounced growth of enterococcalstrains. Strains of other genera than enterococci grewonly as pinpoint colonies on these two agars and couldbe easily distinguished from enterococci. The evaluation(BHI as non-selective medium) yielded productivity valuesclose to 100 % for all three media. Under restrictiveincubation conditions (24 h, aerobic conditions) enterococcalcolonies could be easily distinguished on ECSAagar, whereas the evaluation of the small enterococcalcolonies was more difficult on CATC and mE agar.
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