University of Chicago, Chicago, Illinois Received for publication June 6, 1955

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1 TELLURITE-GLYCINE AGAR: A SELECTIVE PLATING MEDIUM FOR THE QUANTITATIVE DETECTION OF COAGULASE-POSITIVE STAPHYLOCOCCII EUGENE ZEBOVITZ, JAMES B. EVANS, AND C. F. NIVEN, JR. Division of Bacteriology, American Meat Institute Foundation, and the Department of Microbiology, University of Chicago, Chicago, Illinois Received for publication June 6, 1955 The coagulae-positive staphylococci comprise a very homogeneous, readily identifiable species (Evans and Niven, 1950; Shaw et al., 1951) that includes all of the proven pathogenic and enterotoxin-producing strains of staphylococci. As a result of the clinical and public health significance of these organisms, a selective medium for their isolation and enumeration would have considerable value. Chapman (1945, 1946, 1948) has proposed several media that are selective for staphylococci and have some value in differentiating between coagulase-positive and coagulase-negative strains. The selective action of these media is based on a high sodium chloride content and the use of mannitol as an energy source. Their differential value is based on orange pigmentation, acid production from mannitol, and gelatin hydrolysis by coagulase-positive strains. However, not all such strains have these characteristics, and numerous coagulase-negative strains posse some or all of these characteristics. Thus it is frequently necesary to isolate a considerable number of pure cultures from these media and apply the coagulase test to these isolates. A more selective medium has been proposed by Ludlam (1949). This medium includes tellurite and lithium chloride as selective agents and the ph of the medium was adjusted to 9.6 before sterilizing. This medium was found to be quite satisfactory for the isolation of coagulasepositive staphylococci from air, skin surfaces, the nose, and stool specimens (Duguid and Wallace, 1948; Ludlam, 1949). However, Chapman (1949) and McDivitt and Hussemann (1954) 1 Journal paper No. 116, American Meat Institute Foundation. This investigation was supported in part by a research grant from the National Institutes of Health, Public Health Service, and constituted a portion of the thesis submitted by the senior author in partial fulfillment of the requirements for the Ph.D. degree, University of Chicago. found Ludlam's medium to inhibit a significant proportion of the colonies from a coagulasepositive culture. A large number of metabolic inhibitors have been tested in our laboratory as possible constituents in a selective or differential plating medium for the quantitative detection of coagulase-positive staphylococci. As a result of these studies a selective medium has been developed that appears to give results superior to any medium previously available. This medium, tellurite-glycine agar, is derived from that of Ludlam, but includes a number of modifications in composition and in the method of utilization. MATERIALS AND METHODS The composition of tellurite-glycine (TG) agar is listed in table 1. The medium is autoclaved at 15 pounds pressure for 15 minutes, cooled to approximately 50 C, tellurite added aseptically, and poured into sterile petri dishes (20 ml per dish). The surface of the solidified agar is dried by overnight incubation in an inverted position. The surface plating technique of Snyder (1947) is employed in which 0.1-ml aliquots are spread uniformly over the surface of the agar by means of a sterile glass rod bent in the shape of a hockey stick. Graduated 0.2-ml pipettes are employed to measure the 0.1-ml volumes. After inoculation, the plates are incubated in an inverted position at 37 C for 24 hours. Coagulasepositive staphylococci produce black colonies. Further incubation up to 48 hours or longer may allow coagulase-negative staphylococci to grow and produce similar black colonies. It is advisable to prepare the basal medium fresh before each use. If the prepared basal medium is stored in flasks and bottles and reheated to melt the agar, growth of coagulasepositive staphylococci may be inhibited. However, once the TG agar has been poured into petri dishes these may be stored for as long as

2 1955] SELECTIVE MEDIUM FOR COAGULASE-POSITIVE STAPHYLOCOCCI 687 TABLE 1 Composition of tellurite-glycine agar Constituent Per cent in Final Medium Tryptone Yeast extract Mannitol K2HP Lithium chloride Glycine Agar Potassium tellurite (added aseptically after sterilization) The ph is adjusted to 7.2 before autoclaving. days in a sealed container in the refrigerator without loss of selectivity. Potassium tellurite from various commercial sources differs in selective activity as well as in solubility and general appearance of the crystals. We have used potassium tellurite obtained from the Coleman and Bell Company, Norwood, Ohio, for most of this work. A 1.0 per cent aqueous stock solution of tellurite may be sterilized by autoclaving at 15 pounds pressure for 15 minutes. Pure cultures of staphylococci used in this investigation were from the collection of such organisms maintained at the American Meat Institute Foundation. The medium has also been used to detect coagulase-positive staphylococci in foods implicated in food poisoning, in the nose, on the skin, in fecal specimens, in air, and in soil. RESULTS Effect of potassium teuurite. Preliminary experiments as illustrated in table 2 indicated that potassium tellurite at a level of 0.02 per cent was most satisfactory. However, the potency of different lots of tellurite was found to vary over a rather wide range, and these results refer only to the batch of tellurite that has been used throughout most of the work reported here. Effect of glycine. As shown in table 2, a level of 1.0 per cent glycine (in the absence of tellurite) also gave a satisfactory differentiation of the two test strains. However, when other strains were tested it was found that some coagulase-positive strains were slightly inhibited while 6 of 10 coagulase-negative strains grew quite well. Effect of sodium chloride. The effect of increased levels of sodium chloride on the selective activity TABLE 2 Effect of tellurite and glycine on growth of a coagulase-positive (strain 210) and a coagulase-negative (strain HIOA) Staphylococcus Strain Tellurite Quantitative Count on 10' Dilution Concentration No 1.0% 2.0% 3.0% glycine glycne glycme glycine 210 None H1OA None * The basal medium was the same as that given in table 1 but without tellurite, glycine, or lithium chloride. Surface plating was used and incubation was for 24 hours at 37 C. of tellurite and glycine is indicated in table 3. Salt at levels that do not inhibit staphylococci appeared to increase the inhibitory action of teliurite but to decrease the inhibition due to glycine. It also seemed to increase the toxicity of the combination of teliurite and glycine. Evaluation of tellurite-glycine agar. Separate experiments indicated that 0.5 per cent lithium chloride did not alter the selectivity of tellurite and glycine. The medium described in table 1 thus appeared to be the medium of choice. To more critically evaluate this medium 10 coagulase-positive and 12 coagulase-negative strains were selected. These included the strains that in previous experiments had presented the greatest difficulty with these inhibitors. The results of an experiment comparing teliurite-glycine agar with Chapman-Stone agar and phenol red mannitol salt agar are presented in table 4. These platings were made in triplicate and the data represent the average value. The TG agar partially inhibited some of these selected coagulase-positive strains, but generally no more than did the other media. The majority of the coagulase-negative strains were suppressed by the TG agar but not significantly by the high salt media. The colonies produced by strains 9-8 and S-35 on TG agar were small and gray as compared to the larger black colonies produced by coagulme-positive strains. However, the

3 688n ZEBOVITZ, EVANS, AND NIVEN [vol. 70 Strin TABLE 3 Effect of sodium chloride, tellurite, and glycine on growth of a coagulase-positive (strain 210) and a coagulase-negative (strain HIOA) Staphylococcus Quantitative Count on 10 Dilution NaCi Concentration 0.02%o glbo5o 1. glycine te.u0te, te'urite 210 None H10A None The medium and procedure were the same as in table 2. colonies produced by strain S-18 were quite imilar to the coagulase-positive strains. The coagulase-positive strains could not be clearly differentiated from some of the coagulae-negative strains on the C-S or PRMS agar on the basis of gelatin hydrolysis, pigmentation, and acid production from mannitol. Factors affecting the seledcvy of tellurieglycine agar. A number of variations in the methods recommended in this publication have been tested and will be briefly summarized. The use of pour plates rather than surface plating requires a 48-hour incubation for the development of black colonies by coagulase-positive strains, and during this period of incubation many of the coagulase-negative strains grow and produce black colonies. As mentioned previously there appears to be considerable variation in the potency of various batches of potassium tellurite, and it is highly desirable to test each batch on organims of known sensitivity. Ludlam (1953) and McDivitt and Hussemann (1954) both mentioned considerable variation in the apparent potency of different batches of tellurite media. The ph of TG agar is adjusted to 7.2 before autoclaving. Chapman et al. (1937) and Ludlam (1949) recommended the adjustment of ph to 9.6 before autoclaving media for the selective isolation of staphylococci. This may result in TABLE 4 Comparison of the selective action of tellurite-glycine(tg)agar, Chapman-Stone(C)agar, and phenol red mannitol salt(prms)agar on a elected group of staphylococci Quantitative Count on 101 Dilution Strain Coagulase Control TG CS PRMS medium agar aj ar ES J32A H1OA HIOB H16Bt H17C S-19t H2Ct S-35t Pour plates were used for the control medium and the PRMS agar and incubation was for 48 hours at 37 C. Surface plating was used for TG agar and CS agar and incubation was for 24 hours at 37 C. t All counts on these strains refer to the 10- dilution. considerable variation in results (e. g., Chapman, 1949, reported that the final ph of Ludlam's medium was 8.6, while McDivitt and Husemann's 1954 version of Ludlam's medium had a final ph of 9.2). Adjustment of the ph of TG agar to 9.2 before autoclaving increased its inhibitory action on coagulase-positive strains and lengthened the required incubation time to 48 hours. The effect of storage of TG agar under different conditions was also investigated. It was found that if the basal medium (without teilurite) was sterilized and stored in flasks or bottles and then melted before use, it was more inhibitory with respect to coagulae-positive strains. If the

4 1955& SELECTIVE MEDIUM FOR COAGULASE-POSITIVE STAPHYLOCOCCI 689 freshly prepared medium was poured into petri dishes and stored in cans in a refrigerator at 2 C, there was little change in selectivity after as long as 30 days. However, longer storage seemed to result in decreased selectivity. Application of tellurite-glycine agar to the detection and isolation of coagulase-positive staphylococci from natural sources. A limited survey was conducted to determine the effectiveness of TG agar in detecting coagulase-positive staphylococci under conditions where they may be greatly outnumbered by a wide variety of other microorganisms. The medium appeared to be quite satisfactory for all sources studied, including air, dust, soil samples, numerous food products, the skin nose, throat, and fecal specimens. Phenol red mannitol salt agar was used as a control medium. Colonies considered to be typical of coagulase-positive staphylococci were isolated from both media and observed for morphology, gram reaction, and coagulase production. All such isolates from TG agar proved to be positive, and in no instance were coagulasepositive strains isolated from PRMS agar and not from TG agar. Of some interest was the finding in this survey that coagulase-positive organisms were of relatively rare occurrence in air, dust, soil, food products, the skin (other than finger tips), throat, and feces. They were quite common in the nose, and on the fingers of people who harbored them in their nose. The air and dust in an animal room containing chickens were heavily contaminated with these organisms. This medium has also been used in the examination of 16 food samples allegedly involved in cases of food poisoning. Six of these samples were found to contain large numbers of coagulasepositive staphylococci. The TG agar provided a quantitative detection of these organisms. In no case was there any indication from the direct microscopic examination of the samples or plating on other media that TG agar had failed to detect such organisms when they were present. DISCUSSION Tellurite-glycine agar seems to be clearly superior to other selective media currently in use for the quantitative detection of coagulasepositive staphylococci, particularly from sources that may contain substantial numbers of coagulase-negative staphylococci. It is more selective and results are obtained after 24 hours rather than the 48 hours required by other methods. The major short-comings of this medium are, (1) its partial inhibition of some coagulasepositive strains, (2) its failure to inhibit all coagulase-negative strains, (3) the necessity for using a surface-plating technique, and (4) variations in the apparent potency between different lots of tellurite. However, when used as directed it is effective under the conditions of practical usage. The medium is similar to that proposed by Ludlam for the same purpose. It differs in three important features: (1) the use of glycine as well as tellurite as a selective agent, (2) adjustment of the ph to 7.2 rather than 9.6, resulting in less variation as a result of small differences in the time and temperature of autoclaving, and (3) use of the surface-plating technique to give more rapid and clear-cut results. The relative scarcity of coagulase-positive staphylococci in the various natural sources examined is at variance with the rather prevalent misconception that such organisms are ubiquitous. However, it is in agreement with the results of careful experiments such as those of Duguid and Wallace (1948). They reported that subjects regularly yielding heavy growth of these organisms from the anterior nares usually had a few such organisms on the chin, hands, and clothing, but none on the arms, legs, or chest. SUMMARY A medium (tellurite-glycine agar) has been developed that is superior to other media currently in use for the quantitative detection of coagulase-positive staphylococci. This medium contains 1.0 per cent tryptone, 0.5 per cent yeast extract, 0.5 per cent mannitol, 0.5 per cent K2HPO4, 0.5 per cent lithium chloride, 1.0 per cent glycine, 2.0 per cent agar, and 0.02 per cent potassium tellurite (added aseptically after sterilization). The ph is adjusted to 7.2 before autoclaving. A surface-plating technique is employed. Coagulase-positive staphylococci produce black colonies within 24 hours at 37 C. Other microorganisms fail to produce visible growth within this time, with the exception of occasional coagulase-negative staphylococci that may grow. Most of these latter strains produce small gray colonies that are distinct from the type of colony produced by most positive strains.

5 690 ZEBOVITZ, EVANS, AND NIVEN [VOL. 70 This medium has been found satisfactory for the detection of coagulase-positive staphylococci in a variety of food products, fecal samples, soil, air, dust, and on the skin and mucous membranes. REFERENCES CHAPMAN, G. H., LIBB, C. W., BERBNs, C., AND CuRco, L The isolation of probable pathogenic staphylococci. J. Bacteriol., 33, CHAPMAN, G. H The significance of sodium chloride in studies of staphylococci. J. Bacteriol., 50, CHAPMAN, G. H A single culture medium for selective isolation of plasma-coagulating staphylococci and for improved testing of chromogenesis, plasma coagulation, mannitol fermentation, and the Stone reaction. J. Bacteriol., 51, CHAPMAN, G. H An improved Stone's medium for the isolation and testing of food poisoning staphylococci. Food Research, 13, CHAPmAN, G. H Comparison of Ludlam's medium with staphylococcus medium 110 for the isolation of staphylococci that clot blood. J. Bacteriol., 58, 823. DUGUID, J. P. AND WALLACE, A. T Air infection with dust liberated from clothing. Lancet, 1, EVANS, J. B. AND NIVEN, C. F., JR A comparative study of known food poisoning staphylococci and related varieties. J. Bacteriol., 59, LuDLAM, G. B A selective medium for the isolation of Staphylococcus aureu from heavily contaminated material. Monthly Bull. of the Ministry of Health, 8, LUDLAM, G. B Incidence and penicillin sensitivity of Staphylococcus aureu in the noses of infants and their mothers. J. Hyg., 51, McDivirr, M. E. AND HUSSEMANN, D. L Comparison of three media for the isolation of enterotoxigenic micrococci. Am. J. Public Health, 44, SHAW, C., Sqrrrr, J. M., AND COWAN, S. T Staphylococci and their classification. J. Gen. Microbiol., 5, SNYDER, T. L The relative errors of bacteriological plate counting methods. J. Bacteriol., 54, Downloaded from on September 14, 2018 by guest