FLEMING et a1.-BACTERIAL CONTAMINATION I X FOOD YEAST 651

USE OF ANTIBIOTICS IN MEDIA FOR ASSESSING BACTERIAL CONTAMINATION IN FOOD YEAST*

By MURIEL FLEMING, N. H. BARNARD and L. A. ALLEN

A selective medium was required which would permit accurate enumeration of bacterial contaminants in an actively growing culture of food yeast (taken, for example, from a fer- menter). The necessary criteria were that the medium should allow unrestricted growth of species of bacteria likely to be encountered as contaminants in factory conditions, and that the growth of food yeasts should be inhibited by low concentrations of the antibiotic. These criteria were fulfilled by a basal medium of trypbne-Yeastrel-glucose agar in which was incorporated one of the following antibiotics in the concentration indicated : candicidin, filipin, fungichromin or rimocidin (5 p.p.m.) or nystatin (15 p.p.m.).

Introduction In the course of experiments on the growth of various food yeasts in small-scale and pilot-

scale fermenters it became desirable to adopt control measures to detect and assess the degree of infection by bacterial contaminants. A number of antibiotics are now known which are active against fungi while permitting growth of bacteria and media incorporating antibiotics of this character have been described by previous workers. Thus Green & Gray1 recommended a tryptone-Yeastrel-glucose agar medium containing 4 p.p.m. of actidione for suppressing growth of brewer’s yeast, which appears to be particularly sensitive to this antibiotic. Beech & Carr2 made a survey of 26 antibiotics and 20 other organic compounds in their search for selective media for use in examining the mixed microflora of apple juices. Actidione in a concentration of 500 p.p.m. inhibited many, though not all, species of yeast.

Preliminary experiments showed that actidione was much less effective against some of our species of yeast than it was against Sacch. cerevisiae. The present investigation arose in the course of an attempt to find an antibiotic which was active against the yeasts being studied and which could be incorporated in a general medium for routine control.

Experimental The investigation is divided into three parts : (i) selection of a basal medium which would

permit growth of the different groups of bacteria likely to be encountered as contaminants ; (ii) selection of antibiotics able to repress growth of yeasts, while permitting growth of bacteria ; (iii) tests of the selective efficiency of media which incorporated the chosen antibiotics.

Selection of a geqaeral nutrient medium Five species of bacteria, representing a fairly wide range of growth requirements, were used

in testing the potentialities of a general nutrient medium. These were : Bacterium coli (Type I), Streptococcus faecalis, Lactobacillus acidophilus, Lactobacillus casei and Bacillus subtilis.

Two alternative media, likely to permit growth of a wide range of bacterial types, were selected for preliminary tests. These were tryptone-Yeastrel-glucose agar (T.Y.G.A.) and heart- infusion-Yeastrel-glucose agar (H.I.Y.G.A.). T.Y.G.A. is essentially the medium used by Green & Gray1 except that the glucose content was reduced from 5.0 to 0.5% and no indicator was included. The unadjusted pH value is 5-5 ; counts were made on the unadjusted medium and on the same medium after adjusting the pH value to 6.9. H.I.Y.G.A. consisted of heart infusion broth to which was added peptone 1-oy0, NaCl 05y0, Yeastrel 0.3Y0, glucose 0.5% and agar 2.0%.

Colony counts of the test organism on each of these media were compared with those on a control medium, known from results of previous work to be suitable for the growth of that species of bacterium (e.g., a lactose-peptone agar for Bact. coli ; a glucose-Yeastrel-phosphate agar for Str. faecalis; Briggs’ improved medium for lactobacilli ; no control medium was used for aerobic spore-bearers) .

The tests indicated that T.Y.G.A. a t pH 6.9 was likely to prove a satisfactory basal medium in which to incorporate antibiotics to test for their selective action.

* Read a t joint meeting of Microbiology Group and Society of Applied Bacteriology, on 14 January, 1959

J. Sci. Food Agric., 10, December, 1959

The pH value of the final medium was 6.8.

652 FLEMING et a1.-BACTERIAL CONTAMINATION I N FOOD YEAST

Antibiotics and yeasts investigated The following nine antibiotics were selected for testing, partly on account of their antifungal

characteristics, published in the scientific literature, and partly because they were known to be available from either commercial or private sources : actidione, amphotericin B, antimycin A, ascosin, candicidin, filipin, fungichromin, nystatin and rimocidin. All are produced by species of Streptomyces. Actidione is cycloheximide, antimycin A has some of the properties of a nitro- genous phenol ; the remaining antibiotics are polyenes and are rather unstable, particularly in the presence of light.

The yeasts subjected to test were : Saccharomyces cerevisae, Torulopsis uti l is , Candida arborea, Zygosaccharomyces lactis and Rhodotorula gracilis. All these species are edible but they differ considerably in other characteristics. The culture of Sacch. cerevisiae used, a strain of baker's yeast, was included mainly for purposes of comparison with the remaining four species, each of which has some claim to be regarded as a food yeast. T. utilis, which utilises pentoses as well as hexoses and can therefore be grown on waste waters from the paper industry, is the most widely known in this category, although C . arborea has been used fairly extensively, particularly in Germany. 2. lactis ferments lactose and has been grown on dairy wastes. Rh. gracilis develops on normal media as an ordinary protein yeast ; when starved of nitrogen, but provided with an ample supply of air and a suitable source of carbohydrate, it forms large quantities of fat.

Preparation of media containing antibiotics Candicidin and actidione are soluble both in water and in alcohol, amphotericin B is in-

soluble in either liquid, and the remaining antibiotics are either soluble, or give a colloidal suspension, in a mixture of alcohol and water. The general procedure in preparing solutions of these substances, therefore, was to weigh 20 mg. of the antibiotic aseptically in a sterile weigh- ing bottle, add I or 2 ml. of absolute alcohol and make up the resulting solution or suspension to 20 ml. with sterile distilled water. The initial concentration of antibiotic obtained in this way was equivalent to 1000 pg. per ml. Suitable dilutions were prepared from this primary solution or suspension so that, on adding a small measured quantity of each dilution to test- tubes containing 15 ml. of the molten agar medium at 50°, concentrations of the antibiotic ranging from o to 100 pg. per ml. of medium were obtained.

The media containing the antibiotics were poured into Petri dishes and, after the agar had set, the surface of the medium was dried by incubating the plates for one hour at 37O. Suspen- sions of the test yeasts were then streaked across the surface of the agar and the plates transferred to the 30' incubator.

Methods of assessing activity of antibiotics against yeasts Two methods of assessment were adopted : I. The C u p Plate Method.-Large Phoenix* plates, 12 in. square, were used instead of Petri

dishes and into each plate was poured a nutrient medium in which a suspension of the test yeast had been uniformly distributed. After the agar had set, ' cups ' of medium were removed aseptically at suitable intervals in rows and columns over the whole plate.

Solutions containing suitable concentrations of each antibiotic (e.g., 12.5-100 pg. per ml.) were prepared and two drops placed aseptically in the appropriate cup, using for the purpose the standard platinum-tipped dropping pipette described by Harrison et aL4 Plates were examined after incubation at 30°, usually for 24 h. The size and clarity of the zones of inhibition were a rough measure of the effectiveness of the antibiotics against the yeast under test.

l h e Streak Culture Test.-A suspension of a young culture of a yeast was streaked across the surface of the T.Y.G.A. medium, containing a known concentration of the antibiotic under test, in a Petri dish. Five parallel streaks in the same dish were prepared from the five species of yeast. A control plate containing no antibiotic was streaked at the same time. The plates were incubated at 30' and examined after 2 days and subsequently at intervals to see the extent to which growth of each streak had been inhibited by the antibiotic in the medium. Different concentrations of each antibiotic (usually ranging from nil to IOO pg. per ml. of medium) were tested in separate Petri dishes.

* Obtained from British Heat Resisting Glass Co., Bilston, Staffs.

11.

J. Sci. Food Agric., 10, December, 1959

FLEMING et a1.-BACTERIAL CONTAMINATION I N FOOD YEAST 653

An exactly similar set of plates was prepared with Wickerham's medium5-a synthetic agar medium containing glucose, mineral salts and vitamins-in order to test an observation made by Strong.6 He noted that, on a complex medium containing peptone and yeast extract, antimycin A did not inhibit growth of Sacch. cerevisiae, whereas on a simple synthetic medium growth was completely inhibited by quite low concentrations of the antibiotic. It seemed possible, therefore, that a complex medium such as T.Y.G.A. might interfere with the intrinsic inhibitory powers of some of the antibiotics being tested in the present work.

Results The cup-plate method was adopted primarily because it gave a more complete picture of the

comparative activity of different antibiotics against one species of yeast on one agar plate. It proved to be useful in determining major differences in growth-inhibitory power, either by the same antibiotic for different yeasts or by different antibiotics for the same species of yeast. Thus, actidione was effective in a low concentration against Sacch. cerevisiae, it had no action against 2. lactis in the concentrations tested, and was effective in differing degrees in inhibiting the growth of other species of yeast.

There were differences, not only in the width but also in the clarity of the zones and in the sharpness of definition. With C. arborea, for example, the zones of rimocidin, fungichromin and filipin were sharply defined, whereas the zones of actidione were more diffuse in character. This difference is probably inherent in the properties of the molecules of the antibiotics, some diffusing into the agar more rapidly than others. Thus, in the more diffuse zones some yeast cells are able to grow in the peripheral areas. Mere size by itself, therefore, is not a valid criterion of the effectiveness of an antibiotic. Clarity and sharpness of definition should also be taken into account.

Results for Sacch. cerevisiae, 2. lactis and C. arborea are illustrated in Fig. I and Table I. Antimycin A was not included in these particular tests.

FIG I -Zones of anhabatton produced by dzffevent antabaotacs agaanst three specaes of yeasts The ' cups ' in each vertical column were dosed with two drops of the concentration of antibiotic shown a t the base of the column

In general, this method is useful mainly as a preli ide or as a confirmatory measure. A more precise indication of the inhibitory power of tic is given by the streak culture technique.

Results obtained by the latter method indicated that 'candicidin, filipin, fungichromin, nystatin and rimocidin were suitable for incorporation in a medium designed to suppress growth of food yeasts. Antimycin A retarded the growth of yeast but did not inhibit it com- pletely in concentrations up to 50 p.p.m., amphotericin B is not readily soluble, and actidione and ascosin have much lower inhibitory powers than the other antibiotics, actidione having little or no effect on growth of 2. lactis, for example, even in a concentration as high as 100 p.p.m.

Observations on the effect of candicidin, ascosin, nystatin and amphotericin B in the two different media supported Strong's contention that inhibitory action tends to be enhanced on

J. Sci. Food Agric., 10, December, 1959 R R

654 FLEMING et al.-BACTERIAL CONTAMINATIOh~ I T FOOD YEAST

Table I Inhibition of yeast growth by antibiotics measured by zones of inhibition on large plates

(diameters of zones in mm.) Zygosaccharomyces lactis - ~ .~ . ~ . ~ ~~ -~ ~ ~ - Candida arborea

~~

Sacch. cerevisiae ___ Antibiotic Concentration of antibiotic (pg./ml.)

~ - -?.

I00 50 25 12.5 100 50 25 12.5 IOO 50 25 12'5

Rimocidin z j S 22 1 9 ' 13 21 S 17 1 j 10 1 7 s 14 1 3 - Candicidin 22 SD 2 0 19 20 20SD 19 19 18 10 15 SD 14 14 Filipin 20s 18 14 11 18 S 16 13 10 1 6 s 13 12 I 0 + EG + EG Amphotericin B 14 12 12 12 15 14 13 13 II S 10 I 0 I 0 Fungichromin; 18 S 15 1 j - 18 S 1j 13 10 15 S 14 I2 I1

35 33 30 30VD 26VD 24VD 15 VD - - _ _ Actidione 40 Ascosin I 4

A 246 + EG Nystatin 24 SD 22 17 11 VD 2 7 V D 2 2 17 14 22 SD 20 18 15

- - - I O S D - - 13 10 - I0 -

S = Sharp edge to zone EG = Enhanced growth round edge of zone

SD = Slightly diffuse edge to zone VD = very diffuse edge to zone

a synthetic medium. on the complex T.Y.G.A. as on Wickerham's medium.

The remaining antibiotics, on the other hand, appear to be as effective

Results obtained with the five most effective antibiotics are illustrated in Fig. 2 .

FIG. 2.-Strenk cultura tests to assess activity of different antibiotics in inhibiting growth of jive species of yeast

The figures underneath each pair of Petri dishes denote the concentration of antibiotic incorporated in the medium I. T . ufilis 2. C. arborea 3. 2. Inctzc

5 . Sacch. cevevisiac 4. Rh. gracilis

J. Sci. Food Agric., 10, December, 1959

FLEMING et a1.-BACTERIAL CONTAMINATION I N FOOD YEATS 655

Effect of antibiotics on colony counts of bacteria In order to determine whether the five antibiotics found to be most effective against food

yeasts were likely to inhibit growth of contaminating bacteria, which they would be used to detect and enumerate, comparative colony counts were made with the five species of bacteria mentioned earlier. A suspension of each test organism was plated on T.Y.G.A. containing no antibiotic (as control) and on the same medium containing each of the five antibiotics in turn. The con- centration of the latter was chosen so that it would be ample to inhibit growth of food yeasts, so far as could be judged from the results of the foregoing tests-i.e., 5 p.p.m. for candicidin, filipin, fungichromin and rimocidin and 15 p.p.m. for nystatin.

Results (Table 11) showed that none of the antibiotics had an appreciable inhibitory effect on any of the bacteria tested.

Table I1 Effect of antibiotics on colony counts* of bacteria

Antibiotic Concn., Bact. coli Str. faecalis L. casei L. acidophilus B. subtilis p.p.m.

Control none I00 I00 I00 I00 100 Candicidin 5 96 I01 80 I00 97 Filipin 5 80 I02 89 107 88 Fungichromin 5 109 95 83 85 85

88 98 94 97 I00 79 I05 81

Nystatin I5 87 Rimocidin 5 93

* In order to make all the counts comparable, those on plates containing antibiotics have been re-calcu- Each figure is the average of duplicate lated as yo of the counts on the corresponding control plates.

determinations.

Tests with selective media for counting bacterial Contaminants The selective efficiency of media containing suitable antibiotics was tested on samples

withdrawn from the contents of a Ioo-litre fermenter during production of food yeast in a pilot plant. The growth medium used in the pilot plant was refinery cane molasses to which supple- mentary sources of nitrogen and phosphate were added. Serial dilutions of a suspension of bacterial ' contaminants ' were plated on T.Y.G.A. containing no antibiotic. This gave a measure of the number of contaminants actually present. The same dilutions were then plated on T.Y.G.A. in which an antibiotic had been incorporated and to which a I : 100 dilution of the fermenter contents had been added. This gave a measure of the number of contaminants detected by the selective medium in the presence of a large number of cells of food yeast. With each test the fermenter contents alone were plated on T.Y.G.A. to demonstrate the presence of numerous yeast cells, and on T.Y.G.A. plus antibiotic to show that the latter completely suppressed their development into colonies. Plates were incubated at 30" or 37" for 2-4 days, depending on the species of bacteria concerned.

Results of nine tests, involving different combinations of antibiotics, species of food yeast and species of bacterial contaminant (Table 111), showed that an efficient selective medium could be made with any one of the five antibiotics.

Table I11 Results of tests for selective eficiency of T.Y.G.A. medium coetaining a n antibiotic Test Food yeast Antibiotic used Contaminant

Number Numbe; No. in medium Species present* detected

I C. arborea Candicidin Bact . coli 122 I11

2 C. arborea Candicidin Str. faecalis 753 131 3 T. utilis Nystatin L. acidophzlus 46 45

5 T. utilis Nystatin B. subtilis 90 98

7 C . arborea Rimocidin Esch. coli 256 231 8 Z . lactis Fungichromin Str. faecalis 176 167 9 T. utilis Filipin Mixed flora? I97 191

4 T. utilis Nystatin L. casei 67 61

6 C. arborea Rimocidin B. subtilis 81 101

* ' Number ' refers to the colony count, at the same dilution in each test ( I O - ~ , 10-' or 10-8) t From diluted molasses after incubation

J. Sci. Food Agric., 10, December, 1959

656 BARNES-SELECTIVE MEDIA FOR FAECAL STREPTOCOCCI

Conclusions Antibiotics which are active against yeasts and not against bacteria differ appreciably in

the facility with which solutions can be prepared, in their potency against a particular species of yeast and in the concentrations required for efficient selective action in a nutrient agar medium.

It has been shown that a selective medium, which reveals the presence of bacterial con- taminants and enumerates them with reasonable accuracy in the presence of large numbers of food yeast cells, can be prepared by incorporating any one of five antibiotics in a basal medium of tryptone-Yeastrel-glucose agar.

Acknowledgments The authors wish to express their thanks to the Directors of Tate & Lyle Ltd. for permission

to publish these results ; to Mr. D. G. Harrison for preparing the photographs ; and to the follow- ing firms for kindly supplying small quantities of antibiotics for experimental purposes : Boots Pure Drug Co. and Glaxo Laboratories Ltd., in this country ; Commercial Solvents Corp., Chas. Pfizer & Co. Ltd., S. B. Penick 82 Co. and the New York Quinine and Chemical Works Inc., the Squibb Institute for Medical Research and the Upjohn Co., in the United States.

Tate and Lyle Laboratories Keston, Kent

Received 7 April, 1959

References Green, S. R., & Gray, P. P., Wallerstein Labs. Harrison, E., Lees, K. A,, & Wood, F., Analyst,

Covnmun., 1950, 13, 357 1951, 76, 696 2 Beech, F. W,, & Carr, J , G,, J . gen. Micyobiol,, 1955, Wickerham, L. J.8 u.s. Dept. Agric.* '95'> Tech.

Bull. No. 1029

1958 (London: Chapman & Hall) 12, 85 6 Strong, F. M., ' Topics in Microbial Chemistry ',

Briggs, M., J . Dairy Res., 1953, 20, 36

DIFFERENTIAL AND SELECTIVE MEDIA FOR THE FAECAL STREPTOCOCCI*

By ELLA M. BARNES

Both sodium azide and thallous acetate have been incorporated separately in media for the selective isolation of the faecal streptococci (Lancefield group D). It has been found that the various methods developed for increasing the selectivity of the sodium azide media often eliminate Sty. bovis. The importance of this in relation to these organisms as indicators of faecal pollution in water and foods is discussed. By the addition of tetrazolium to thc thallous acetate agar a t pH 6.0, it is possible to distinguish colonies of Sty. faecalis and variants zylnogenes and liquefaciens from Sty. faecium and Sty. durans. This method has been used for determining the numbers and types of faecal streptococci in the intestines of the various meat animals and poultry and to show changes in species following antibiotic feeding.

Introduction Before a discussion of differential and selective media for the faecal streptococci, it is neces-

sary to define the characteristics of this group. The term ' faecal streptococcus ' is usually confined to those organisms which are serologically of Lancefield group D and have the following properties : they are catalase-negative, facultatively anaerobic cocci which are able to grow in the presence of 40% bile, hydrolyse aesculin and produce acid from lactose. They are normally

J. Sci. Food Agric., 10, December, 1959

* Read a t joint meeting of Microbiology Group and Society of Applied Bacteriology, 14 January, 1959