MYGP+COPPER, A MEDIUM THAT DETECTS BOTH SACCHAROMYCES AND NON- SACCHAROMYCES WILD YEAST IN THE PRESENCE OF CULTURE YEAST*

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1 J. hist Brew.. May-June, 98. Vol. 90. pp. - MYGPCOPPER, A MEDIUM THAT DETECTS BOTH SACCHAROMYCES AND NON- SACCHAROMYCES WILD YEAST IN THE PRESENCE OF CULTURE YEAST* By G. T. Taylor a A. S. Marsh (Courage Brewing Limited. Imperial Way. Reading RG 0PS) Rnvnvd June /v.s'.t Lin's copper sulphate medium for the detection of non-saccharomyces wild yeast was modified, mainly by reduc ing the concentration of copper sulphate, with the aim of producing a medium that could detect a wide range of both non-saccharomyces a Saccharomyces wild yeast. The performance of the resultant MYGP Cu medium was compared to that of crystal violet a media using pure cultures of wild yeast, pitching yeast a brewery samples. The growth of the pitching yeasts examined was suppressed more in MYGP Cu than in crystal violet a the ale yeasts were more susceptible to copper inhibition than the lager yeasts. Neither MYGP Cu nor crystal violet could detect all the wild Saccharomyces species tested but the proportion detected by each was similar. Twenty-seven out of 8 non-saccharomyces wild yeasts tested could be detected in MYGP Cu. When brewery samples were examined, the detection rate by MYGP Cu compared favourably with that by a combination of crystal violet a. MYGP Cu is particularly suited to the monitoring of the production of cask-coitioned ales. Key words: brewers yeast, contamination, detection, method, saccharomyces. wild yeast. Introduction Wild yeasis are hazardous brewery contaminants, particu larly of cask-coitioned beers. They are generally detected by plating in selective media that enhance their growth rela tive to that of culture yeasts. Rapid methods of detection such as immunofluoresccnt staining6"0"" have been developed but they have not been sufficiently advantageous to replace plating methods in routine use. However plating methods are not entirely satisfactory a improvements have been sought. One major improvement would be to produce a single medium that supports the growth of all wild yeasts whilst suppressing the growth ofculture yeasts. The present media"6" te to detect mainly cither Saccharomyces wild yeasts, or non-saccharomyces wild yeasis. Saccharomyces species of wild yeast arc generally detected in crystal violet medium.' Schwartz differential medium, Lin's crystal violet/fuchsin sulphite medium." modified crystal violet medium" a modified Lin's medium* are alternatives to crystal violet. Non-Saccharomyces species of wild yeast arc generally detected on medium.*""' This medium will also detect some species of Saccharomyces that do not grow in crystal violet." A disadvantage of medium is that it can support a slight development ofculture yeast. Alter natives to are the recently formulated Lin's copper sulphate medium* a actidione medium." which when supplemented with calcium carbonate can be used to detect Brettanomyccs. Lin's copper sulphate medium was formulated to have a concentration of copper sulphate that would suppress the growth of both culture yeast a most other Saccharomyces species. However the work published showing the develop ment of the medium suggested that some Saccharomyccs wild yeast may be more resistant to copper than culture yeasts. Thus an attempt was made to modify Lin's copper sulphate medium so that both Saccharomyccs a non- Saccharomyces wild yeast could be delected in one medium. During the course of this work, Rocken" published the details of pantothenate agar that was developed with a similar aim. Methods Media The new selective medium was based on MYGP (YM- ) agar which contains per litre distilled water: malt 'Pan of this work was presented at the British Yeast Group Mod ing, 6/ January 98, Queen Mary College, Loon. extract ( g). yeast extract ( g). glucose (0 g). bactopeptone ( g) a agar (0 g). The medium was steamed to melt the agar. dispensed into 0ml portions a autoclaved at I C for minutes. A copper sulphate solution (0.000 ppm CuSO) was auto claved separately a the required amount was added to the medium immediately before pouring the plates. Autoclaving copper sulphate in the medium caused browning. Crystal violet medium containing 0 ppm crystal violet a % agar was prepared from hopped pale ale wort clari fied by filtering previously autoclaved wort. The crystal violet was added dissolved in 9% ethanol ( ml/litre medium) after the medium had been steamed to melt the agar. The medium was dispensed a autoclaved as above. Calcium carbonate medium contained per litre distilled water: glucose (0 g), calcium carbonate ( g), malt extract ( g), yeast extract ( g), bactopeptone ( g), actidione (00g), thiaminc HC (00 g) a agar (0g). The medium was steamed, dispensed a autoclaved as above. medium () was prepared according to the manufacturer's instructions except that it was autoclaved at 08 C for 0 minutes in 0 ml portions. Yeasis The yeasts from the Courage culture collection that were examined are listed in Table I. The names arc according to Lodder" a previous names are shown in brackets. The list of Saccharomyces wild yeasts includes. cerevisiae a S. uvarum strains which may be similar to culture years. Several non-saccharomyces species from a variety of sources were examined including yeasts (D98 to DI) that are unable to ferment. The non-characterized yeasts had been isolated from the brewery environment a some had been named tentatively. Yea si Growlh All incubations were at C. Yeasts were cultured from a small inoculum in double strength MYGP broths (0 ml) contained in McCartney bottles. After days growth they were stored in a refrigerator until required. Yeasts were diluted in J strength Ringer's buffer prior to plating by the pour plate method. When the ability of crystal violet, a media to grow wild a culture yeast was compared, 0 ml of the MYGP culture of the yeast a 0 ml of a I0 to 0~s dilution were plated in these media a in MYGP. The MYGP control was used to check that the recovery of the yeasts in the selective media was satisfac tory. In general growth on the plates was recorded after days a I week incubation. When these media were com pared using brewery' samples, the plates were incubated for - days.

2 ^ Vol.90, 98] TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS TABLE I. Yeast strains examined (a) Culture yeast Ale. cerevisiae A, A, A, A0 Lager S. itvarum B, B, B6 (b) Saccharomyces a non-saccharomyces species Courage culture collection number Name National collection of yeast cultures number Source D6 D0 D D D D D8 D9 D60 D68 D9 D9 D0 DI6 D D9 S. cerevisiae (S. cerevisiae var. ellipsoideus) S. italicus S. rouxii S. italicus (S. steineri) S. bisporus var. mellis (S. mellis) S. bayanus (S. oviformis) S. bayanus (S. pastorianus) S. uvarum (S. validus) S. cerevisiae (S. cartilis) S. bayanus (. oviformis) S. bailii S. bayanus S. bailii var. bailii l*y etegansy S. cerevisiae (S. turbidans) S. cerevisiae (S. cerevisiae var. turbidans) S. cerevisiae (killer yeast) Chianti grape must Cane sugar Melrose & Drover Wine yeast Long Ashton Research Station Long Ashton Research Station Draught beer Draught beer Continuous fermentation plant Dll D D6 D9 D D6 D D D0 D8 D9 Caida utilis Hansenuta anomala (variety not known) Pichia membranefaciens Debaromyces hansenii (D. kloeckeri) Brettanomyces lambicus Saccharomycodes ludwigii Torulopsis sphaerica imperfect form of Khuyveromyces lactis Hansenula anomala Saccharomycodes ludwigii Kloeckera apiculata Schizosaccharomyces pombe Lambic beer Wine yeast Beer Cider Brewing yeast Cider Long Ashton D9 D96 D9 D98 D D0 D0 D0 D0 D06 D0 D08 DI09 DUO Dill - Dl D Brettanomyces bruxellensis Brettanomyces anomalus Brettanomyces anomalus Debaromyces hansenii Pichia membranefaciens Trigonopsis variabilis Cryptococcus terreus Cryptococcus albidus var. aiffluens Caida humicola Hansenula canadensis Nematospora coryli Rhodotorula marina Pichia quercuum Debaromyces vanriji Caida muscorum Rhodosporidium diobovatum* Hansenula wingei English brewery, stout Stout Spoiled beer (stout) Cheese Draught beer Soil Soil Soil Frass from Pinus resmosa Hazel nuts Shrimp Slime flux of Quercus kcllogii Roots of oak seedlings *Name as NCYC catalogue

3 6 TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS [J. Inst. Brew. (c) Non-characterised brewing isolates Courage culture collection number Tentative name Source a description D D D D D8 D6 D6 D6 D8 D6 D D8 D D0 D D D Saccharomyces diastaticus Saccharomyces diastaticus Brettanomyces sp Rhodotorula rubra Caida sake S. cerevisiae S. cerevisiae S. cerevisiae Produces ethyl acetate in beer Produces ethyl acetate in beer Superannuating Wild yeast ex culture yeast Wild yeast from bottle-coitioned ale, very attenuative Isolated from hazy a superannuated ale. Osmophilic yeast ex syrup Film forming wild yeast isolated ex ale con ditioning tank Isolated from bottle-coitioned stout Isolated from ale with estery/glue aroma Isolated ex cask-coitioned ale Isolated ex complaint sample Isolated ex beer main Isolated ex cask-coitioned ale Isolated ex cask rackcr Isolated ex cask-coitioned ale TABLE II. Inhibition of ale a lager yeasts by varying concentrations of copper sulphate Yeast count after plates incubated for days Yeast count incubated after plates for days Yeasi A0 B6 A0 B6 Dilution plated [CuSO] ppm I0" io-«i0" JO" io- io-«0-' 0-" 0 Inn Inn Inn Inn 0 Inn 9 Inn ,,,0 Inn = Innumerable Results MYGP was chosen as the basal medium. It can be easily prepared or purchased complete from. The sup plement of ammonium chloride used by Lin" was not added because MYGP agar gives good yeast growth without sup plementation of the nitrogen source. Likewise, the supple ment of dipotassium hydrogen phosphate was not added because it influences the inhibition by copper sulphate ofthe growth of culture yeast." Lin reported" that the coitions of pre-storage of the copper sulphate plates prior to use as spread plates influenced the degree of yeast inhibition. This source of variation was eliminated by the use of pour plates with the medium being treated with copper sulphate immediately before plating. The minimum concentration of copper sulphate that would inhibit the growth of culture yeasts in MYGP agar was determined (Table II). 0 ppm copper sulphate inhi bited the growth of an ale yeast a 80 ppm copper sul phate inhibited the growth of a lager yeast. Those yeast cells that survived the lower concentrations of copper sulphate developed into 'dot-sized' colonies a the number that survived depeed on the number of cells plated. The effect of changing the supplier of the ingredients of the MYGP medium was examined. MYGP agars prepared with several combinations of materials were used to deter mine the concentration ofcopper sulphate that was required to inhibit the growth of the ale yeast (Table HI). The effect of the copper sulphate was partially neutralised in media containing agar obtained from BDH (media a 6). More over these media were cloudy whereas those prepared with agar were clear. Changing the supplier of the other ingredients had less effect on the copper toxicity except that the prepared from YM medium (medium ) was slightly more inhibitory. Medium I was used in all further trials. A concentration of copper sulphate of ppm (- mm Cu) was chosen for the medium so that some dilution by the volume of sample could be tolerated. The influence of the nature of the sample to be plated upon the suppression of the growth of the culture yeasts was examined (Table IV). Water, Ringer's buffer, pasteurised beer or clarified, autoclaved, brewery wort were added to a plate containing 0- ml of a suspension of culture yeast in water a mixed with 0 ml agar. The number of yeast colonies that developed was compared to the number of yeast plated as determined by growth in MYGP. The ale yeast was completely inhibited by even

4 Vol. 90, 98] TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS TABLE III. Effect of ingredient supplier on inhibition of growth of culture yeast by copper sulphate """""" "^^Code Componcnr~~~~^^^ 6» Yeast extract Malt extract Peptone Glucose Agar BDH BDH BDH BDH BDH BDH All components except agar supplied as YM Broth. Yeast count after plates ncubated for days Yeast count after plates incubated for days Yeast A0 AO Dilution plated io- 0-" Code 6 6 [CuSOJppm S TABLE IV. Effect of the inoculation medium on the suppression of the growth of culture yeasts Incubation time for the plates of yeast cells Growth in medium (0 ml) with additions Yeast (days) plated None Water Ringer's buffer Pasteurised beer Autoclavcd wort lml ml lml ml lml ml ml ml A 0 0 0,000 8 <0- mm 9 <0-mm 09 <0-mm B6 0,000 (<0-- mm) (<0-- mm) 0 (<0-- mm) (0-- mm) Colony sizes shown in brackets. in the presence of % (v/v) beer or wort. The inhibition of the lager yeast was not affected by the addition of water or Ringer's buffer. However, the addition of beer or wort did permit a slight development of the lager yeast. The colonies were very small after days incubation but their size continued to increase upon exteed incubation. Care would have to be taken when interpreting the results of plating brewery samples containing lager yeast, particularly if large numbers were present (Table II). This difficulty could be overcome by restricting the volume of wort or beer plated or by removing the yeasts by centrifugation a resuspeing them in water prior to plating., crystal violet a media were com pared for their ability to support the growth of the wild yeasts a some culture yeasts in the Courage culture collection. In order to assess the effect of inoculum size upon growth, both the neat broth culture a a KH-lfJ- dilution were plated in the media a in MYGP as a control (Table V a, b, c, d). The amount of growth a the maximum colony size were recorded after days a week incubation. The results are listed in Table V (a, b, c, d) a summarised in Table VI. When a large inoculum of culture yeast was plated (Table Va), there was some development in all selective media. With all the culture yeasts except B, sup ported less growth than crystal violet or. When a small number of cells (lo'-lo) was plated, neither nor crystal violet supported growth. supported a

5 8 TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS [J. lnst. Brew. limited amount of growth a this property can make the interpretation of plates difficult. All but three of the Saccharomyces species of wild yeast grew in (Table Vb). The yeasts that did not grow were S. uvarum (D9), S. cerevisiae (formerly. cartilis) (D60) a S. cerevisiae (formerly S. cerevisiae var. turbidans) (DI). D60 a D grew in crystal violet but D9 did not grow in either crystal violet or, which makes it iistinguishable from culture yeast. The growth of one other yeast (D9) was significantly less in MYGP Cu than in crystal violet. However most of the Saccharomyces wild yeasts, including another strain of 'S. turbidans', pro duced as much growth or more in as in crystal violet. Six yeasts namely strain of S. rouxii (D), strains of S. bayanus (D, D8 a D68) a strains of S. bailii (D9I a D0) did not grow in crystal violet but did grow in. Contamination by of these yeasts may be detectable with the conventional media because the yeasts develop to some extent in the medium but contami nation by the other yeasts (D a D9) would not be detectable with the conventional media. All the non-saccharomyces species grew in both a (Table Vc). Nine yeasts (D, D6, D0, D9, D9, DI0, D0, D08 a Dl ) grew poorly in but some of these (D9, D0, D0 a Dl ) a also D8S grew in crystal violet. Yeasts DI9, DI09 a Dl grew to a lesser extent in than in the con ventional media ( for D9 a DI09, crystal violet for Dl ) a the growth of DI09 in was variable. The growth of the remaining yeasts in was either equal to or better than that in a crystal violet. The Brettanomyces species, D9, D96 a D9, grew slowly in both a so that an exteed incuba tion period would be required to detect these yeasts. The same restriction applies to the calcium carbonate/actidionc medium that is used to detect Brettanomyces. With the non-characterised yeasts, two (D a DI0) did not grow in a others (D a D8) grew better in crystal violet or than in (Table Vd). The growth of the remaining yeasts in MYGP Cu was equal to or better than that in crystal violet or. Yeast D produced only small colonies in a a it did not grow in crystal violet. It may be possible to discern this yeast from the culture yeast in conventional media but it should be easier in. Some of the yeast species (8%) produced brown colonies in (Table VI). This should aid the enumeration of these colonies in routine plating. The yeasts were grouped according to whether growth in was significantly better than, or about the same as, or worse than that in crystal violet or (Table VI). The efficiency of the media to enumerate several wild yeasts from each group was then compared (Table VII). Between 0 a 00 wild yeast cells together with (-) x 0} ale yeast or lager yeast cells were plated in medium a in crystal violet or as appropriate. The number of discernable wild yeast colonies that developed was expressed as a percentage of the number developing in the MYGP control without culture yeast. Any interference by the culture yeast in the enumeration would have increased the apparent recovery of wild yeast. There was a variable backgrou development of the lager yeast throughout this test a it may have interfered slightly in the wild yeast count with D9 a D6. However, D9 (Brettanomyces bruxellensis) is slow-growing a so the plates had to be recorded at days rather than or days. In most cases the wild yeast count appeared to be unaffected by the culture yeasts. As expected from Table Vb, the Saccharomyces yeasts, D, D8 a DI0, were not detectable in crystal violet. D8 a D0 could be enumerated in but all yeasts were detected in. was more efficient than for the detection of D8 because the wild yeast colonies in were difficult to discern from those of the culture yeast. was less efficient than (a) Culture yeasts TABLE V. Comparison of crystal violet, a for supporting growth of wild a culture yeasts Yeast Dilution plated Neat (i) lo)-v)o'(ii) Incubation time days week days week Medium A AU A A B B B6.... <0- (j) Growth on MYGP control was taken as. (ii) Colony counts in MYGP not shown.

6 Vol.90,98] TAYLOR AND MARSH: MEDIUM TOR DETECTION OF WILD YEASTS 9 (b) Saccharomyces wild yeast Yeast Dilution plated Neat lo-vio- Incubation time days week days week Medium D < <0-mm D0 6 - <0-6 - <0- D D - 9 <0-9 = <0- D 0 - < <0- D <- < D D9 <0-0- D60-0- = D D9I 9 <0- < D9 * <0 6 <0- D0 0 9 <0- < DI DI D Recovery of D9 in variable.

7 0 TAYLOR AND MARSH: MEDIUM KOR DETECTION OF WILD YEASTS [J. Inst. Brew. (c) Non-Saccharomyces wild ycasl Dilution plated Neat I0-VI0" Yeast Incubation time days week days week Medium Dll 6-6 D 9-9 DI6 MYGP Cu <0-0 D9 < D = <0-66 <0- D6 98 <0-98 D < D D < D D <0- <0- <0S <0- D9 ~* =0- D96 <0-8 - = 0- D9 8 <0-6 = -

8 Vol.90,98] TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS (c) Non-Saccharomyces wild yeast (continued) Dilution plated Neat 0-VlO-' Yeast Incubation time days I week days week Medium D98 9 <0- < DIOO DI0 6 0 <0- <0- <0-6 6 I <0- DI I D0 D D D0 - D I D09 * 9 < DUO Dill - Dl D 9 8 < *- - Recovery of D09 in variable =not determined.

9 TAYLOR AND MARSH: MEDIUM KOR DETECTION OF WILD YEASTS [J. Inst. Brew. (d) Non-characterised brewery isolates Dilution plated Neat io-v 0' Yeasi Incubation time days week days week Medium D D 0-0 DI D D8 0 8 <0- - < D6 9 < <0 D6 0 <0-0 8 = D <0-0 0 ~ -- <0- D D = - D8 0 < T- DI 9 0 <0- D DI 0 8 > D DI

10 Vol. 90, 98] TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS TABLE VI. Summary of growth responses of the wild yeasts shown in Table V Saccharomyces species Non-Saccharomyccs species Non-characterised yeasts Growth in better than in crystal violet or DI.D. D68. D9I.DIO D6, D. D6, D0, D9, D96, D9. DI06. DI08 D6, D8. DI6. DI Growth in same as in crystal violet or D6, D0. D. D. D8, DI6. DI9 DI.D. D. D. D8. D9. D98, DIOO. D0.D0. DI0. DI0. DO,DIIO,DI,DI D. D. D6.D6. DI, D8. DII.DI. DI Growth in worse than in crystal violet or D9. D9, D60, D DI9. D09, Dl D, D, D8. DUO No appreciable growth in D9. D60. D D. DI0 Yeast producing fawn or brown colonies D. D0 D.DI, DI9, D, D. D9, D9, D96, D9. D98. D, DI0, DI0. DI0. D09, DII D8, D6, D8, DI8, D TABLE VII. Comparison of with crystal violet or for enumeration of wild yeasts in the presence of culture yeasts % Recovery in MYGP Cu % Recovery in crystal violet or Expected efficiency ranking of the media Wild yeast - Ale yeast Lager yeast Medium Ale yeast Lager yeast Growth in better than in crystal violet or Saccharomyccs D DI Non-Saccharomyces D6 D9 DIOI Non-characterised D8 Dl 6 DI Growth in same as in crystal violet or Saccharomyccs D6 D0 D D Non-Saccharomyces D D Growth in worse than in crystal violet or Non-characterised Saccharomyces Non-Saccharomyces D D6 D8 D60 D9 D9 D Non-characterised D D Plates counted after days a week incubation (a also days for D9) crystal violet for the detection of yeasts (D8 a D9). Three yeasts (DI, D60 a DI09) were not detected by even though the results in Table Vc suggested that DI09 would be detected. With the remaining yeasts, the detection rates in the media were similar, irrespective of the initial ranking of the efficiency of the media (Table VI). The performance of the medium merited an assessment of its ability to detect wild yeasts in routine microbiological quality control samples. The detection rate ofwild yeasts in medium was compared to that in crystal violet a media (Figures la a b). The majority of the samples plated were cask-coitioned beers but several samples of'beer in process' in the Development Brewery, of bottle-coitioned stout a of brewery pitching yeast were also plated. The plating results shown in Figures la a b are those in which a significant number of wild yeasts was detected. The most frequent contaminants were presumptive Saccharomyces wild yeasts that grew in crystal violet. These yeasts were readily detected in. In some cases the count in was greater than that in crystal violet or [e.g. arrow A in Figure la) or there was a small count in medium but none in crystal violet or (e.g. arrows D, E, F). A colony from the plate of samples A, D, E, F was isolated, grown in MYGP broth a re-plated in MYGP,, crystal violet a. The yeast from sample A grew in the wild yeast media, although growth

11 TAYLOR AND MARSH: MEDIUM FOR DETECTION OF WILD YEASTS {J. Inst. Brew. A s E s n (b) C DEF lljl a I o S o I..,.. jjh Crystal Violet S too s I..... II....I...L..III.I,ill. i.l. i.i.i, I!I. II. I. Inlhlllllil....I.I..I.I.. Pig.. Comparison of wild yeast counts in, crystal violet a MYCPCu. Vertical bas show the number of wild yeast detected in a sample with each medium. in crystal violet was less than that in a. The yeasts from samples E a F grew equally well in crystal violet a, even though they were not detected initially in crystal violet. The yeast from sample D grew in a even though it was not detected initially in. The growth of this yeast in both a was slow a the colony size in did not exceed that expected for culture yeast until after days incubation. These yeasts were isolated from pitching yeast a it was concluded that the greater suppres sion of the growth of the pitching yeast in enabled them to be differentiated from pitching yeast in but not in crystal violet or. The sample denoted by the arrow C in Figure lb was a bottle-coitioned stout. Stouts are plated in a calcium carbonate medium to detect Brettanomyces species. Sample C contained many Brettanomyces (> lovml) a this con tamination was not detected in medium, crystal

12 Vol.90, 98) TAYLOR AND MARSH: MHDIUM POR IJKTIiCTION Oh WILD YKASTS violet or above a low level of contamination by olhcr wild yeasts, probably because of the slow growth rate of the yeast. Yeasts were detected in the crystal violet plate of sample B (Figure Ib) but not in themygp Cu plate. When a yeast colony from the crystal violet plate was grown up a replated, it grew in both MYGP Cu a in crystal violet. The reason for the failure to detect this yeast initially in was not elucidated. Sample G gave an appreciable count in that was not accompanied by a similar count in MYGP Cu, even though the isolated yeast grew equally well in MYGP Cu a. Sample G contained many bacteria a it was inferred that the growth of these bacteria in the initial MYGP Cu plate prevented the growth of the wild yeast. Although brewery bacteria can develop in MYGP Cu, it was fou during the assessment that bacterial colonies could be readily distinguished from those of wild yeasts. Discussion Microbiological quality control is still based on the results of plating samples, despite the inherent delay. Each medium used for plating has its limitations. Those media that rely on the selective inhibition of culture yeasts do not detect all the wild yeasts within their inteed range."" medium a pantothenate-frce agar rely on a nutrient requirement for selectivity"" a so are liable to interference by nutri ents in the sample. Moreover medium contains suf ficient nutrient to allow a slight development of the culture yeasts, particularly of those plated in low numbers." The main advantage of the medium is that it can selectively support the growth of a wide range of wild yeasts, even when brewery samples arc plated. The effective ness of the copper sulphate inhibition of culture yeasts might be expected to be reduced by materials in brewery samples such as divalent ions a natural cheiatingagents!(u but this was fou not to be a serious problem. The inhibition was affected by the type of agar used in the medium. The cells of culture yeast that survived in the MYGP Cu media con taining low concentrations of copper sulphate (Table II a III) may have had an increased copper-resistance. Copperresistance can be iuced in S. cerevisiae by progressive sub-lethal selection, but this was not a problem in the fully effective medium. does not detect all Saccharomyccs wild yeast but neither do crystal violet. Lin's crystal violct/fuchsin sulphite or Schwarz differential media.*-8"8 The culture yeast studied developed less readily in than in 0ppm crystal violet medium or medium. a the other media share the limitation that they allow the growth of bacteria but with experience colonies of bacteria can be distinguished from those of yeast. The medium performed well in brewery trials, aiding the investigation of a case of contamination by Saccharomyces wild yeast. 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