Differential Agar Medium for Separating Streptococcus

Transcription

1 APPLED MICROBIOLOGY, Nov. 1969, p American Society for Microbiology Vol. 18, No. 5 Printed in U.S.A. Differential Agar Medium for Separating Streptococcus lactis and Streptococcus cremorist M. SRINIVASULU REDDY, E. R. VEDAMUTHU, C. J. WASHAM, AND G. W. REINBOLD Department of Food Technology, Iowa State University, Ames, Iowa Received for publication 19 August 1969 The characteristic ability of Streptococcus lactis and inability of Streptococcus cremoris to hydrolyze arginine formed the basis for the development of a differential agar medium to separate these species in pure and mixed cultures. Ammonia liberated from arginine was detected by the ph changes occurring in the medium. The agar contained milk as the sole source of carbohydrate, arginine as the specific substrate, diffusible (K2HPO4) and nondiffusible (CaCO3) buffer systems, and a suitable ph indicator in addition to other ingredients. The nondiffusible buffer system afforded the localization of ph changes, and, hence, the indicator color changes immediately around individual colonies appearing on the medium. S. cremoris produced yellow colonies surrounded by yellow zones on this purple medium because of their ability to produce acid from lactose in the milk. S. lactis, on the other hand, first produced colonies similar to S. cremoris, but subsequent color reversal of ph indicator with the liberation of NH3 resulted in the discharge of the yellow color. Hence, S. lactis colonies were white and devoid of zones. The difference in their colony color allowed the identification of the species in a mixture of S. cremoris and S. lactis strains. The medium was found suitable for both qualitative and quantitative differentiation. The most widely used microorganisms in dairy starters belong to Sherman's lactic group of streptococci (15) classified under serological group N (14). Extensive studies on this group have been made by Orla-Jensen (12) and Sherman (15). Recently, Sandine et al. (13) made a comprehensive study of the cultural characteristics of this group. Ayres et al. (2) were the first to study systematically the production of NH3 by streptococci and point out the value of this characteristic as an aid in differentiating species within this genus. Hills (7) showed that some streptococci could stoichiometrically liberate 1 mole each of NH3 and CO2 from 1 mole of arginine. Niven et al. (11) then devised a suitable test medium for arginine hydrolysis and demonstrated its value in species differentiation within the lactic group of streptococci. They found that Streptococcus lactis hydrolyzes arginine invariably and Streptococcus cremoris generally does not. The arginine hydrolysis test broth has since then been widely used to separate S. lactis and S. cremoris. On the basis of this biochemical characteristic several attempts have been made to devise differential media for these organisms. Turner et al. (16) 1 Journal Paper no. J-6340 of the Iowa Agriculture and Home Economics Experiment Station, Ames. Iowa; project no developed an agar medium incorporating L-arginine and 2,3,5-triphenyltetrazolium chloride, which separated S. cremoris and S. lactis by the color difference of the colonies; the former produced white and the latter red colonies on this agar. The authors, however, pointed out that the ph of the medium was quite critical for obtaining consistent results and that variable results were obtained with certain strains of the two species. Mikolajcik (10) described an arginine broth that could be used to differentiate S. crernoris from S. lactis in pure cultures; it was, however, unsuitable for mixtures of these two species. In all the attempts cited here, the differentiation was based upon the detection of NH3 liberated from arginine. This was accomplished in the test proposed by Niven et al. (11) by reaction with Nessler's reagent and in Mikolajcik's broth (10) by incorporation of a ph indicator which changed color as the ph of the medium shifted toward the neutral or basic side, liberating NH3. In the agar medium of Turner et al. (16), the reduction of 2,3,5-triphenyltetrazolium chloride was inhibited by the acid produced from glucose, but when the acid was neutralized by the ammonia from arginine, the suppressive effect of acid was reversed. The incorporation of Nessler's reagent in an agar medium would com- 755

2 756 REDDY ET AL. APPL. MICROBIOL. pletely prevent the growth of microorganisms because of its high toxicity (8); hence, it is inapplicable. The main drawback in the use of a ph indicator lies in the inability to prevent diffusion of acidic or basic byproducts throughout the agar, resulting in an overall color change of the entire medium toward the acidic or basic range. Because of this, differentiation in mixed cultures containing both S. cremoris and S. lactis strains is impossible. Recently, Keston et al. (9) described an agar medium to differentiate Escherichia coli colonies (developing on the same plate) capable and incapable of producing acid from sugars. In this medium, the acid produced by any colony is localized around it by homogeneously suspended CaCO3, which acts as a nondiffusible buffer. In our investigations, this technique along with several modifications of Mikolajcik's broth (10) were applied to develop a suitable differential medium for S. cremoris and S. lactis. MATERIALS AND METHODS Cultures. Ten strains of S. cremoris and of S. lactis were used in this study. The cultures were obtained from the collections at the Department of Food Technology, Iowa State University; Department of Microbiology, Oregon State University; and from D. B. Emmons, Food Research Institute, Canada. The cultures were maintained by tri-weekly transfer in reconstituted 11.0% Matrix milk (Galloway- West Co., Fond du Lac, Wis.) and incubated at 30 C for 18 hr. Between transfers, they were stored at 5 C Ṁedia. The differential medium contained 0.5% tryptone, 0.5% yeast extract, 0.4% L-arginine hydrochloride, 0.1% K2HPO4, 0.3% CaCO3, 0.6% carboxy methyl cellulose (CMC Cekol, MV, Uddeholm, Sweden), and 1.5% agar. Before pouring plates, 5.0 ml of sterile 11% reconstituted nonfat dry milk, and 2.0 ml of sterile 0.1% BCP (bromocresol purple) in distilled water were added to 100 ml of agar; the amount of agar necessary to make up 1.0 liter of the medium was suspended in 500 ml of distilled water and steamed until dissolution. In another glass beaker containing 500 ml of distilled water, 6.0 g of CMC was suspended and heated in a boiling water bath until the opacity disappeared. The two portions were mixed together in a stainless-steel container with the required quantities of tryptone, yeast extract, arginine, K2HPO4, and CaCO3, covered with aluminum foil, and steamed for 10 min. The ph of the medium after steaming should be 6.8 i The agar was then dispensed into bottles in 100-ml quantities and sterilized at 121 C for 15 min. The other ingredients (nonfat milk and BCP) were added (just before pouring plates) as described earlier. The CMC in the medium facilitated the suspension of the insoluble CaCO3 in the medium. For comparative studies on the merits of the differential medium for quantitative recovery of the fastidious streptococci, Eugonagar (BBL, Cockeysville, Md.) was used. Previous work in our laboratory had shown Eugonagar as good as the lactic agar of Elliker et al. (6) for the recovery of lactic streptococci. Plating technique. Agar plates were prepared as follows. After adding the necessary amount of milk and indicator to the melted medium tempered in a 55 C water bath, the bottle contents were mixed thoroughly, avoiding incorporation of air, and poured into previously chilled (at 5 C) petri plates to obtain a layer 4 to 5 mm thick. The chilling of the petri dishes facilitated the rapid gelation of the agar, ensuring homogeneous suspension of CaCO3 in the agar layer. After the medium solidified, the plates were dried for 18 to 24 hr in an incubator at 37 C. Decimal dilutions of the cultures were made according to Standard Methods for the Examination of Dairy Products (1). Samples (0.1 ml) of the dilutions were spread evenly with a bent glass rod on the surface of the agar layer. Wherever more than one strain (or species) were plated together, 1.0 ml of each of the respective dilutions of the strains (species) was mixed together in a sterile screw-cap test tube, and 0.1 ml of the resultant mixture was spread on the agar surface. Conventional pour platings were made according to Standard Methods (1). The plates were incubated in air and in candle oats jar at 32 C and were examined after 48 hr. RESULTS AND DISCUSSION Preliminary studies were conducted in test tubes to establish the optimal levels of the various ingredients in the medium for obtaining the critical color changes of the indicator with the growth of the microorganisms. In these experiments, the medium did not include milk and agar, but contained glucose. After determining the suitable levels, agar was added and plating studies were made. The levels of glucose and arginine in these preliminary trials ranged from 0.05 to 0.5% and 0.3 to 0.5%, respectively. The combination that gave the required indicator color changes was 0.1% glucose and 0.4% arginine. Initially, very poor growth was observed on the plates. To support the growth of fastidious streptococci, the agar was fortified with different levels of skim milk. This enhanced the recovery of the organisms, and, from these trials, the optimal level of milk was established. The addition of milk, however, altered the critical sugar-arginine-buffer balance in the medium, and certain slow acidproducing strains of both species failed to yield a clear-cut differentiation. It was argued that the milk would have enhanced the sugar level, thus altering the balance. On elimination of dextrose and reduction of K2HPO4 level (reduced from 0.2 to 0.1 %), the medium gave excellent growth and differentiation for all possible combinations of the 10 respective strains of S. cremoris and S. lactis. The former produced small yellow colonies

3 VOL. 18, 1969 DIFFERENTIAL MEDIUM FOR LACTIC STREPTOCOCCI 757 with yellow zones and the latter larger white colonies with no surrounding zones on the finalized medium (Fig. 1). This color differentiation was evident in 48 hr and persisted throughout an extended incubation period of several days. When arginine was deleted from the proposed medium, both S. cremoris and S. lactis produced yellow colonies surrounded by yellow zones. Excellent results were obtained when the culture dilutions were spread on the agar with a bent glass rod and incubated in a candle oats jar at 32 C. When conventional pour plating was tried, the differentiation was not as distinct as when spreading technique was used. Candle oats jar incubation enhanced the colony size, and results were consistently available in 48 hr. Air incubation yielded very small colonies and the period required for obtaining the results varied with different strains, the minimum being 4 days. Freshly made medium gave the best results. Table 1 shows the counting efficiency on differential agar of pure and mixed cultures of S. Downloaded from FIG. 1. Photograph showing colonial growth of S. cremoris DR7 (left), S. lactis C2F (middle), and mixture of the two (right) on the differential agar medium. TABLE 1. Plate countsa offive S. cremoris and five S. lactis strains in pure cultures and representative mixtures and their colony color on the differential agar Colony color Pure cultures Mixed cultures S. cremoris S. lcuis S. cremoris S. lactis Stiain S. I strainb strainb strainb strainb mixture c remoris a S. c t s colonies/ml colonies/ml colonies/ml colonies/ml A Yellow F White A 0.5 X 108 F 14.0 X 10 A-G 0.8 X X 108 B Yellow G White B 7.2 X 108 G 7.2 X 108 B-G 6.6 X X 108 C Yellow H White C 1.4 X 108 H 18.0 X 108 B-I 6.8 X X 108 D Yellow I White D 2.6 X 108 I 14.0 X 108 D-H 2.8 X X 108 E Yellow J White E 11.0 X 108 J 26.0 X 108 E-H 11.0 X X 108 a By spreading technique and after incubation in candle oats jar at 32 C for 48 hr. b A, Sc3; B, Scl; C, HP; D, W; E, DR7; F, 10; G, C2S; H, E; I, 11454; J, The letters code for the corresponding strains of S. cremoris and S. lactis. Strain mixtures were made as described in the text. on December 13, 2018 by guest

4 758 REDDY ET AL. APPL. MICROBIOL. TABLE 2. Efficiency of recovery of S. cremoris and S. lactis strains on differential agar as compared to Eugonagar Colonies per mib Strain" Agar Surface spread Pour plating Airc COJC Airc COJC A Differential 7 X 101 Eugonagar 7 X X X X 108 B Differential 9 X 10, Eugonagar 11 X X 10, 12 X X 108 C Differential 7 X 108 Eugonagar 8 X X X X 108 D Differential 7 X 108 Eugonagar 7 X X X X 108 E Differential 19 X 10 Eugonagar 19 X X < X 108 F Differential 17 X 1081 Eugonagar 18 X X X X 108 A-Dd Differential 7 X 108 (3 + 4) ex 108 Eugonagar 8 X X X X 108 B-Ed Differential 14 X 108 (4 + 10)e X 108 Eugonagar 16 X X X X 108 C-Fd Differential 11 X 108 (3 + 8)e X 108 Eugonagar 11 X X X X 108 a A, S. cremoris Scl; B, S. cremoris DR7; C, S. cremoris MIA; D, S. lactis C2S; E, S. lactis E; F, S. lactis Incubation at 32 C for 48 hr. c Environment of incubation; COJ, abbreviation for candle oats jar. d Strain mixtures were made as described in the text. e Figures in parentheses indicate the counts of the individual strains in the twin mixture. The first figure represents the count of S. cremoris and the second the S. lactis strain in the mixture. cremoris and S. lactis strains. The differential recovery of the organisms from the mixtures was excellent for all possible combinations of the strains shown in Table 1. In some instances, however, when one of the strains in the mixture had a very high initial count at plating, that strain tended to dominate and accurate counts of the less numerous strain were not obtained. In such instances, when higher dilutions of the former were plated with lower dilutions of the latter, better differentiation and recovery were possible. The efficiency of recovery of fastidious streptococci by differential medium as compared with Eugonagar is shown in Table 2. In this experiment with Eugonagar, both surface spread and pour plating techniques were used. Duplicate platings with Eugonagar were incubated in air and candle oats jar at 32 C to study the effect of incubation environments on the colony counts. The results in Table 2 indicate that the differential agar provided as good recoveries as Eugonagar. The plating technique and the incubation environment did not affect the counts on Eugonagar. These results indicate that the proposed medium and technique could be used both for qualitative and quantitative differentiation of mixtures of S. cremoris and S. lactis strains. Four strains of Streptococcus diacetilactis were also tested on this medium. All formed large

5 VOL. 18, 1969 DIFFERENTIAL MEDIUM FOR LACTIC STREPTOCOCCI 759 white colonies similar to S. lactis. Currently, investigations are in progress to modify the medium to allow the differentiation of all three species on the same plate. The differential medium offers many advantages to the dairy industry and starter culture manufacturers. The tedium of species differentiation of mixed strain starters by picking a large number of individual colonies into a separate broth to screen for the hydrolysis of arginine is eliminated. Further, if large cheese operations with laboratory facilities want to check periodically the species mixture in commercial cultures used in their operation, this could be done rapidly and economically. This could be very important because S. diacetilactis and certain strains of S. lactis are considered undesirable for Cheddar cheese manufacture (17, 18). In addition, investigations on compatability and dominance in mixed strain starters would be feasible by use of the differential agar (19). Modifications of this medium could also be used in the study of slow acid-producing variants, mutants of S. cremoris and S. lactis incapable of utilizing lactose in enzymatic studies involving metabolism, and acid production among these organisms (3, 4, 5). Deletion of arginine, addition of 0.2% lactose (added as a filter-sterilized solution), and substitution of milk by another growth stimulant yielded an excellent medium for the detection of lactose-negative and slow acidproducing variants of S. cremoris and S. lactis even on crowded plates. LITERATURE CITED 1. American Public Health Association, Inc Standard methods for the examination of dairy products, 12th ed., p American Public Health Association, Inc. New York. 2. Ayres, S. H., Rupp, P., and Mudge, C. S The production of ammonia and carbon dioxide by streptococci. J. Infec. Dis. 29: Citti, J. E., Sandine, W. E., and Elliker, P. R. 1965a. Comparison of slow and fast acid-producing Streptococcus lactis. J. Dairy Sci. 48: Citti, J. E., Sandine, W. E., and Elliker, P. R. 1965b. 0- Galactosidase of Streptococcus lactis. J. Bacteriol. 89: Citti, J. E., Sandine, W. E., and Elliker, P. R Lactose and maltose uptake by Streptococcus lactis. J. Dairy Sci. 50: Elliker, P. R., Anderson, A. W., and Hannesson, G An agar culture medium for lactic acid streptococci and lactobacilli. J. Dairy Sci. 39: Hills, G. M Ammonia production by pathogenic bacteria. Biochem. J. 34: Hodgman, C. D., Weast, R. C., and Selby, S. M Handbook of chemistry and physics, 42nd ed., p The Chemical Rubber Publishing Co., Cleveland. 9. Keston, A. S., and Rosenberg, D Medium for differentiation of acid-producing colonies with homogeneously suspended calcium carbonate. J. Bacteriol. 93: Mikolajcik, E. M Single broth for the differentiation of Streptococcus lactis from Streptococcus cremoris. J. Dairy Sci. 47: Niven, C. F., Jr., Smiley, K. L., and Sherman, J. M The hydrolysis of arginine by streptococci. J. Bacteriol. 43: Orla-Jensen, S The lactic acid bacteria. 2nd ed. I Kommission Hos Ejnar Munksgaard, Copenhagen, Denmark. 13. Sandine, W. E., Elliker, P. R., and Hayes, Helen Cultural studies on Streptococcus diacetilactis and other members of the lactic Streptococcus group. Can. J. Microbiol. 8: Shattock, P. M. F., and Mattick, A. T. R The serological grouping of Streptococcus lactis (Group N) and its relationship to Streptococcus faecalis. J. Hyg. 43: Sherman, J. M The streptococci. Bacteriol. Rev. 1: Turner, Nikki, Sandine, W. E., Elliker, P. R., and Day, E. A Use of tetrazolium dyes in an agar medium for differentiation of Streptococcus lactis and Streptococcus cremoris. J. Dairy Sci. 46: Vedamuthu, E. R., Sandine, W. E., and Elliker, P. R. 1966a. Flavor and texture in Cheddar cheese. L. Role of mixed strain lactic starter cultures. J. Dairy Sci. 49: Vedamuthu, E. R., Sandine, W. E., and Elliker, P. R. 1966b. Flavor and texture in Cheddar cheese. IL. Carbonyl compounds produced by mixed strain lactic starter cultures. J. Dairy Sci. 49: Vedamuthu, E. R., Hauser, B. A., Henning, D. R., Sandine, W. E., Elliker, P. R., and Day, E. A. 1966c. Competitive growth of Streptococcus diacetilactis in mixed strain lactic cultures and cheese. Proc. 17th Int. Dairy Congr. D(2),