A NEW MEDIUM, SALT MANNITOL PLASMA AGAR, FOR THE ISOLATION OF STAPHYLOCOCCUS AUREUS

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1 TITE AMERICAN JOUBNAL OF CLINICAL PATHOLOGY Copyright 1967 by The Williams & Wilkins Co. Vol. 47, No. 1 Printed in U.S.A. A NEW MEDIUM, SALT MANNITOL PLASMA AGAR, FOR THE ISOLATION OF STAPHYLOCOCCUS AUREUS E. B. BLAIR, PH.D., J. S. EMERSON, AND A. H. TULL, B.S. Microbiology Division, U. S. Army Medical Research and Nutrition Laboratory, Fitzsimons General Hospital, Denver, Colorado During studies of the staphylococcal carrier rate among medical personnel, two selective media, salt milk agar (SMA) and man--j*f nitol salt agar (MSA), were used. Salt milk ' ; agar was unsatisfactory because white Staphylococcus aureus strains were overlooked; also, the enhancement of chromogenesis on this medium resulted in selection of many pigmented, Gram-positive cocci that were coagulase-negative. Mannitol salt agar presented difficulties in early colony differentiation, especially when heavy concentrations of weakly pigmented, acid-producing colonies turned the entire medium yellow (acid); or when a few nonpigmented, weak acid producers were surrounded by large numbers of salt-tolerant colonies that turned the medium alkaline. Attempts to overcome some of the above difficulties resulted in the development in our laboratory of a medium that we have called salt mannitol plasma agar (SMPA). MATERIALS AND METHODS Mannitol salt agar 6 from Baltimore Biological Laboratories (BBL), Baltimore, Maryland, and tellurite glycine agar 12 (TGA) from Difco, Detroit, Michigan, were prepared in accordance with instructions on the label. SMA. 2 To 570 ml. of distilled water, 40 Gm. of BBL trypticase soy agar (TSA) and 70 Gm. of NaCl were added. The medium was heated to dissolve the salt and agar and then autoclaved at 121 C. for 15 min. To the sterile, cooled (65 C.) medium, 430 ml. of heated (55 C.) evaporated milk were added. The final NaCl concentration was 7.5 %. Sheep blood agar (SBA). Defibrinated sheep blood was added to a final concentration of 5 % to sterile, cooled (50 C.) TSA. Received, May 6, The use of commerical trade names is for the purpose of identification only and does not constitute an endorsement by the Department of Defense. SMPA. Trypticase (BBL), 15 Gm.; beef extract (Difco), 1 Gm.; NaCl, 75 Gm.; D-mannitol, 10 Gm.; and bromcresol purple, 4 ml. of a 0.5 % solution, were added to 1000 ml. of distilled water. The reaction was adjusted to ph 6.9, after which 15 Gm. of agar were added. The medium was heated to dissolve the agar, autoclaved at 121 C. for 15 min., and allowed to cool to 50 C. Warmed (37 C.)i coagulase-reactive, outdated human blood bank plasma, 100 ml., was added and thoroughly mixed with the cooled medium. The medium was dispensed to plates in 15- ml. amounts. The final reaction was ph 7.0 ± Preparation of indicator. Dibromo-o-cresol sulfonphthalein NF,* 0.25 Gm., was added to 1S.5 ml. of 0.01 N NaOH. The dissolution of the dye was completed by adding 31.5 ml. of distilled water and autoclaving it at 121 C. for 15 min. The clear stock indicator solution contained Gm. of dye/ml. The SMPA medium was later modified by the addition of 2 ml. of neutral fat emulsion per liter of sterile medium. Satisfactory stable suspensions of oil (soya bean, olive, or cottonseed), prepared with 25% oil and 2% acacia in water, were sterilized by autoclaving after being emulsified in an electric blender or with an ultrasonic probe. The emulsion was stored at 4 C. and shaken vigorously before use to ensure even distribution of larger fat droplets. Deoxyribonuclease (DNase). The DiSalvo 30 method 7 for DNase production was performed by transferring cells from suspect colonies to DNase test agar (Difco) and flooding them with 1 N HC1 after incubation for 24 hr. at 35 C. Square Integrid (100 by 15 mm.) platesf were used, and the culture was touched to the medium in the center * National Aniline Division, Allied Chemical and Dye Corp., 40 Rector St., New York, New York. t Falcon Plastics, 5500 West 83rd Street, Los Angeles, California

2 Jan A NEW MEDIUM FOR S. AUREUS 31 of a square, rather than being streaked, thus allowing 36 tests per plate. During evaluation of the DNase reaction, all suspect colonies were tested further for clumping factor and, if clumping factor was absent, the coagulase test was performed. Later, only DNase-positive isolates were tested further for coagulase or clumping factor. Clumping factor* Five tests were performed on each microscope slide with human blood bank plasma diluted 1:10 with Difco heart infusion broth (HIB). Plasmas were pretested with staphylococci that were weak coagulase producers. Growth from IS- to 24-hr. TSA cultures was suspended in a drop of water, and the suspension, if smooth, was mixed with a drop of diluted plasma. Only immediate clumping was regarded as a positive result. Strains that clumped in water were tested for coagulase. Coagulase test. 6 All suspect colonies negative for clumping factor were subjected to the coagulase test. A loopful of 18- to 24-hr. growth from TSA was mixed in 0.5 ml. of a 1:10 dilution of plasma in HIB. The mixture was incubated in a 37 C. water-bath for 24 hr. Readings were made at short intervals for the first 4 to 6 hr. Any degree of coagulation was recorded as positive. Collection of specimens. Cultures from the mandibular area of the face and the anterior nares were collected on cotton swabs moistened with HIB. Throat specimens were collected on dry cotton swabs from the tonsillar areas of the pharynx. Swabs were immediately inoculated on approximately one-third of the agar plate. For parallel testing of media the specimen was collected on a single swab which was used for inoculation of all media. Excellent colony isolation was obtained by using a dry, sterile swab to carry one-half of the previously inoculated sector, in a clockwise direction, to a new area while the swab was rotated so that a fresh portion was brought into contact each time with the area just inoculated. This method, in addition to being more rapid, resulted in better distribution of the flora, and generally provided more colonies for examination than do other commonly used methods. Incubation of cultures. All cultures were incubated at 35 C. for 24 hr. Culture reactions were noted and suspect colonies were selected, when possible. The plates were incubated at 35 C. for an additional 24 hr. and re-examined. SMA, MSA, SMPA, and SBA plates were maintained at room temperature (22 to 25 C.) for additional periods of up to 10 days to await possible pigment development on these media, and surface plaque and precipitate formation on SMPA. TGA plates were discarded after 72 hr. Selection of colonies. Colonies suspected of being S. aureus were picked from the various media on the basis of: SMA, pigment; MSA, pigment, acid production, bright white appearance, which "bright" characteristic, also seen on SMPA, represented colonies that were nonpigmented and non-acid-producing, but were differentiated from surrounding colonies by a glistening surface and a greater opacity; SMPA, pigment, a blue zone surrounding the colony, clearing around the colony (only on plates containing fat emulsion), precipitate in medium around the colony, surface plaques surrounding the colony, acid production, bright white appearance; TGA, black or gray colonies; and SBA, hemolysis, pigment. RESULTS Efficacy of MSA and SMA for Recovery of S. aureus SMA was the only medium used during the first carrier surveys, and was unsatisfactory, as previously stated. MSA was tested parallelly with SMA in two surveys utilizing nasal and facial swabs. The efficacy of MSA and SMA for recovery of S. aureus from nasal cultures is shown in Table 1. Coagulase-positive staphylococci were found in 86 of the 254 specimens. With both media, 70 specimens yielded positive results, accounting for SI.4% of the total positives. MSA was positive for an additional 13 specimens, whereas parallel SMA plates were negative; conversely, the SMA revealed three positive cultures that were negative on the companion MSA plates. MSA was successful in recovering 96.5% of the total positives from this population, whereas only 84.9 % of the positive specimens were demonstrable on the SMA plate. The discrepancy was probably due to nonselection of white, coagulase-positive colonies on SMA. In each

3 32 BLAIR ET AL. Vol. 4-7 instance in which the SMA plate but not the MSA plate was positive, the number of S. aureus colonies did not exceed four per plate. The facial swabs produced few positive cultures on either medium. The obvious enhancement of pigment production on SMA led to an inordinate amount of work because of the many pigmented, salt-tolerant, coagulase-negative cocci found in these specimens. Correlation of DNase and Coagulase Production It has been reported that occasional strains of coagulase-positive staphylococci failed to produce DNase. 3-8 ' 10 Our studies on 2415 suspect colonies (Table 2) indicated that all 13S5 clumping factor- or coagulasepositive strains (or both) were also DNasepositive; however, 223 isolates (16.1 % of the total DNase-positives) were negative for TABLE 1 EFFICACY OF MANNITOL SALT AGAR AND SALT MILK AGAR (SMA) TO RECOVER S. AUREUS FROM NASAL Medium* MSA+ SMA+ MSA+ SMA- MSA- SMA+ Total CULTURES No./Total Cultures 70/254 (27.6%) 13/254 (5.1%) 3/254 (1.2%) 86/254 (33.9%) (MSA) (No. Positive/Total 1 Positive 70/86 (81.4%) 13/86 (15.1%) 3/86 (3.5%) 86/86 (100.0%) * The positive and negative symbols indicate growth and no growth, respectively, on the media inoculated simultaneously. TABLE 2 DEOXYRIBONUCLEASE AND COAGULASE PRODUCTION OF 2415 NOSE, THROAT, AND FACE ISOLATES DNase and Coagulase Reactions DNase+ Coagulase+ DNase+ Coagulase DNase Coagulase No./Total Tested* 1385/2415 (57.3%) 223/2415 (9.2%) 807/2415 (33.4%) * Isolates from SMA, MSA, and SMPA. clumping factor and coagulase. Therefore, we felt justified in using the DNase test for screening out coagulase-negative colonies but not as a substitute for determination of coagulase. In retrospect, S07 (33.4%) of coagulase-negative colonies could have been eliminated by the DNase test alone. After this study colonies from one specimen exhibited an orange pigment typical of S. aureus on SMPA, but produced a very weak DNase reaction on the DiSalvo medium. Clumping factor and coagulase tests on these colonies were positive and the colonies were phage-typable. Observed Reactions of S. aureus on Salt Mannitol Plasma Agar S. aureus colonies on SMPA were orange, yellow, or bright white; coagulase-negative staphylococci and micrococci were bluish, dull in appearance, or bright white (Fig. 1). S. aureus colonies from approximately 75 % of the positive specimens were surrounded by a blue zone, which has not been defined as yet (Fig. 2). Colonies from relatively few specimens produced sufficient acid to change the indicator, probably as a result of a combination of the effects of the higher pk (S.32) of bromcresol purple, high NaCl content, and the buffering action of the plasma (Fig. 3). Acid-producing colonies did not form blue zones, although after prolonged incubation at room temperature some of the blue zoning colonies produced a weak acid reaction around the colony and within the zone. In addition, within 6 days only about 50 % of the S. aureus had formed a halo of precipitate in the medium surrounding the colony (Figs. 1, 2, and 6), in contrast with the coagulase-mannitol agar of Esber and Faulconer. 9 Precipitate formation in the medium was affected by different plasmas, and also was present when serum was substituted for plasma. Colonies from a majority of the specimens producing blue zones were surrounded by the lipid plaques dedescribed by Weld 11 appearing on the surface of the agar (Fig. 4). These plaques formed on media containing chylous plasma within 24 hr., whereas up to 10 days' incubation was required when clear plasma containing little fat was used. The neutral fat emulsion

4 Jan.1967 A N E W M E D I U M F O R S. AUREUS 33 TTppPpSif 4i/0' "Jl wm FIG. 1 (upper). SMPA without fat emulsion. Nasal culture incubated for 4S hr. at 35 C. and for 24 hr. at room temperature (22 to 25 C ). Dark gray colonies in photograph (actually orange) are S. aureus surrounded by white coagulase-negative organisms. Precipitate appears around the pigmented colonies. White colonics close to <S. aureus colonies occasionally form partial or complete halos of precipitate. FIG. 2 (lower). SMPA without fat emulsion. S. aureus colonies surrounded by: a, zone (blue); and 6, dense white precipitate.

5 34 BLAIR ET AL. Vol. 47 additive also assured early appearance of these plaques. The acid biotypes did not form plaques. Colonies producing a blue zone simultaneously hydrolyzed or broke the fat emulsion, forming a zone of clearing in the turbid plate (Figs. 3, 5, and 6). This zone was shown to correlate with lipase production by the formation of blue insoluble copper soaps around the larger fat droplets in the zones of clearing when the C11SO4 method described by Berry 1 was used. Changes in composition of the medium greatly affected the ability of S. aureus to form either the blue zone or demonstrable lipase. The peptone was found to be important for only two of the eight peptones tested, tryptone (Difco) and N-Z amine type B (Sheffield), which yielded results similar to trypticase (BBL). Variation in concentration of the bromcresol purple indicator caused undesirable changes in the reaction in the medium. Replacement of beef extract by yeast extract resulted in an increased number of acid reactions and a subsequent adverse decrease in blue zone production. S. aureus requirements for biotin, thiamine, and nicotinic acid were satisfied in the medium, as the added vitamins (alone and on combinations) did not enhance growth or improve reactions; in fact, added thiamine resulted in increased acid reactions. Reactions by and Recovery of S. aureus on SMPA and MSA Results of studies undertaken during carrier surveys to determine the efficacy of SMPA (without fat emulsion) and MSA to recover S. aureus from nose and throat cultures are listed in Table 3, and indicate no significant difference between the two media. Both media demonstrated S. aureus in 194 (S6.6%) of the 224 positive specimens. SMPA plates were positive in 12 instances, whereas parallel MSA plates were negative. Conversely, 18 specimens were positive on MSA but not on SMPA. With one exception, the number of S. aureus colonies appearing on the positive plate rarely exceeded four when the other plate was negative, and it was thought that sampling variation probably accounted for these differences. One exception is noteworthy: the SMPA plate demonstrated, through production of the blue zone, an almost pure culture of coagulase-positive staphylococci which were nonpigmented and mannitol-negative, and which, on initial isolation, were nonhemolytic on SBA. The biochemical reactions produced on SMPA by S. aureus recovered from nasal and throat cultures are summarized in Table 4. Colonies producing a blue zone were invariably S. aureus and were found in 211 (76.4 %) of the 276 positive specimens. Pigment was present in 266 (96.4 %) of the total positives within 3 days, and 49 (17.8%) produced sufficient acid in the medium to change the indicator. These studies revealed that 149 (97.4%) of 153 strains producing precipitate in the medium within 6 days were coagulase-positive; however, colonies from 54.0% of the positive specimens produced a precipitate. Of specimens with bright white colonies, 57 yielded only one coagulase-positive Staphylococcus. These will be discussed later. Inasmuch as a single colony may simultaneously exhibit as many as three or four of the described reactions, the effectiveness of categorizing colonies according to individual and multiple reactions in relation to coagulase production is shown in Table 5. Precipitates were excluded because longer periods were required for their formation and they could occur with any of the other reactions. Blue zone or pigment (or both) were formed in 271 of the 276 positive specimens, accounting for 98.2 % of the total positives. Colonies producing acid but no zone or pigment were found in seven specimens, of which four were coagulasepositive and comprised only 1.4% of the total positives. The reactions of acid and pigment production on MSA are shown in Table 6. Coagulase-positive staphylococci from 239 (93.7 %) of 255 positives exhibited an acid reaction in the medium no matter what the pigment was, whereas 10 (3.9%) of the positive specimens were found on the basis of pigment alone. Of all specimens with colonies on MSA or SMPA manifesting one or more of the described characteristic reactions, 249 (8S.0 %) of 2S3 and 275 (S7.3%) of 315, respectively,

6 FIG. 3 (upper). SMPA with fat emulsion. Wound culture. One S. aureus colony surrounded by zone of clearing (lipase). All other colonies are acid biotypes of S. aureus that turn the medium yellow. Phage types of acid and lipase producers were identical: 52/52A/80. FIG. 4 (lower). SMPA with fat emulsion. S. aureus incubated for 4S hr. at 35 C. and for 24 hr. at room temperature (22 to 25 C ). Lipid plaques appear as irregular masses of crystal-like material on the agar surface surrounding the colonies. 35

7 36 BLAIR ET AL. Vol. 47 were coagulase-positive. Therefore, when only these reactions formed the criteria for selection of colonies, the chances for success on either medium were equal. A significant difference between the two media was the ability of SMPA to characterize S. aureus that were nonpigmented and nonreactive on MSA. Colonies chosen on the basis of "bright only," in Tables 4 and 6, were positive in only one (1.8%) of 57 specimens on SMPA, as compared with six (11.5 %) of 52 similar specimens on MSA. The specimen with nonreactive colonies on SMPA has been the only one encountered to date and was also nonreactive on MSA. The remaining five MSA-nonreactive specimens all produced blue zones on SMPA. Therefore, when MSA is used these bright colonies should not be ignored. The few positive colonies that appeared after 48 hr. have not been included in the previous discussion and tables. SMPA exhibited three positive specimens in this category: one, pigment only; one, pigment and zone; and one, bright only; MSA showed six: one, pigment only; three, pigment and acid; and two, bright only. Parallel studies with four media, SBA, TGA, MSA, and SMPA, showed differences in early colony differentiation, although all were equal in ability to recover S. aureus ultimately from nose and throat swabs. Within 48 hr. several positive specimens on SBA were recorded as negative; however, re-examination of these plates revealed nonpigmented, nonhemolytic S. aureus of the same phage type as those on the other media. In addition, it was found that both gray and TABLE 3 EFFICACY OF SALT MANNITOL PLASMA AGAR (SMPA) AND MANNITOL SALT AGAR (MSA) TO RECOVER S. AUREUS FROM NOSE AND THROAT SPECIMENS Number/Total Cultures Nose Throat Total SMPA+ MSA+ SMPA+ MSA- SMPA- MSA+ SMPA- MSA- 153/444 (34.5%) 7/444 (1.6%) 10/444 (2.3%) 274/444 (61.7%) 41/183 (22.4%) 5/183 (2.7%) 8/183 (4.4%) 129/183 (70.5%) 194/627* (30.9%) 12/627* (1.9%) 18/627* (2.9%) 403/627 (64.3%) * Total positives = 224. TABLE 4 INDIVIDUAL CHARACTERISTICS OF S. AUREUS ISOLATED ON SMPA FROM NOSE AND THROAT SWABS Specimens with Suspect Colonies Zone+ Pigment+ Acid+ Precipitate+ Bright only Total No. positive/no. showing reactionf 211/211 (100.0%) 266/303 (87.8%) 49/63 (77.8%) 149/153 (97.4%) lt/57 (1.8%) 276/372 (74.2%) No. positive/total positive 211/276 (76.4%) 266/276 (96.4%) 49/276 (17.8%) 149/276 (54.0%) 1/276 (0.4%) * Maximal time limits: Zone, 48 hr. (35 C); pigment, 48 hr. (35 C.) and 24 hr. (room temperature); acid, 48 hr. (35 C); precipitate, 48 hrs. (35 C.) and 72 hr. (room temperature), t A single colony may exhibit three of the four reactions simultaneously, t Pigment appeared after 5 days.

8 F i o. 5 (upper). SMPA with fat emulsion. T h r o a t culture showing: a, S. aureus colony without a zone of clearing (non-lipase producer); and b and c, two chromogenic types producing clearing (lipase). Phage types: a, 71; b, 6/47/83A; c, nontypable. Fid. 6 (lower). SMPA with fat emulsion. Nasal culture showing S. aureus which are nonpigmentcd and surrounded by zones of clearing (lipase) and white precipitate within the clearing. 37

9 38 BLAIR ET AL. Vol. 47 TABLE 5 EFFECTIVENESS OF COMBINATIONS OF CHARACTERISTIC REACTIONS OF NOSE AND THROAT FLORA ON SMPA TO DEMONSTRATE S. AUREUS Specimens with Suspect Colonies No. positive/no. showing reaction No. positive/total positive Zone+ and/or pigment+ Zone, pigment,, acid+ Bright only Total 271/308 (88.0%) 4/7 (57.1%) 1/57 (1.8%) 270/372 (74.2%) 271/270 (9S.2%) 4/270 (1.4%) 1/270 (0.4%) 270/270 (100.0%) TABLE 0 CHARACTERISTIC REACTIONS OF S. AUREUS ISOLATED ON MANNITOL SALT AGAR FROM NOSE AND THROAT SWABS Specimens with Suspect Colonies Acid + Acid pigment+ Bright, only Total No. positive/no. tested 239/203 (90.9%) 10/20 (50.0%) 0/52 (11.5%) 255/335 (70.1%) No. positive/total positive 239/255 (93.7%) 10/255 (3.9%) 0/255 (2.4%) 255/255 (100.0%)! Maximal time limits: acid, 48 hr. (35 C); pigment, 48 hr. (35 C), and 24 hr. (room temperature). black colonies from TGA must be selected at 24 hr., and black colonies selected at 48 hr. DISCUSSION In our experience, SMPA was far superior to the other media tested with respect to early colony differentiation and identification. Several specimens contained an acid biotype, as well as a few colonies exhibiting lipase production (Fig. 3). The latter were obvious even in the midst of large numbers of acid-producing S. aureus. These metabolic differences between strains from a single specimen were not necessarily reflected in the phage types, as it was found that phage types of the lipase producer and the acid biotype could be either identical or entirely different. Likewise, variation in chromogenesis in a single specimen could not always be associated with differences in phage type. Repeated wound cultures from a patient continued to demonstrate three pigment types that were identical in phage type, whereas a throat culture from another patient on SMPA revealed three different phage types, a pigmented non-lipase producer and two lipase producers varying in chromogenesis (Fig. 5). Elucidation of the phenomena observed in this medium on initial cultures of clinical specimens, and their possible relation to the disease process, may lead to a better understanding of the pathogenesis of S. aureus. For example, SMPA, as well as SBA, revealed the heterogeneity of a lyophilized culture of the 502A strain used to colonize infants. A vial from the same lot that was used for artificial colonizing of newborns was sent to our laboratory for examination after use of this strain resulted in a high incidence of minor lesions among the infants involved. Both media demonstrated three colony types in the lyophile vial used for colonization, as well as in another vial from the same lot. Phage types of these isolates were only suggestive of strain differences. SUMMARY Salt mannitol plasma agar (SMPA) was found to be equal to mannitol salt agar and

10 Jan A NEW MEDIUM FOR S. AUREUS 39 other selective and nonselective media in recovering S. aureus from nose and throat cultures. It was superior to other media with regard to ease of colony differentiation and identification, making SMPA a valuable addition for the diagnostic laboratory. Subtle -etabolic differences between strains within a single specimen were often revealed on SMPA, thus providing an additional research tool for examination of population variation. Acknowledgments. Specialists Fifth Class B. E. Gordon and C. L. Woodley provided technical assistance. Lt. Col. W. A. Paccy, CO., and personnel of the 249th General Hospital, and Major Faith A. Oldham, Director, as well as the personnel of the Advanced Medical Technician Course, Fi Usurious General Hospital, cooperated in conducting carrier surveys. REFERENCES 1. Berry, J. A.: Detection of microbial lipase by copper soap formation. J. Bad,., 25: , Blair, J. E., Jackson, G. G., Kuhn, L. R., and Wise, R. J.: Staphylococcus Infections. In Diagnostic Procedures and Reagents, Ed. 4, Ch. VI. New York: American Public Health Association, Inc., 1903, p Burns, J., and Holtman, D. F.: Biochemical properties of virulent and avirulent staphylococci. Ann. New York Acad. Sc, 88: , Cadness-Gravcs, B., Williams, R., Harper, G. J., and Miles, A. A.: Slide-test for coagulasepositive staphylococci. Lancet, /: S, Chapman, G. H.: The significance of sodium chloride in studies of staphylococci. J. Bact., 50: , Cowan, S. T.: Classification of staphylococci by precipitation and biological reactions. f. Path., 46: 31-45, DiSalvo, J. W.: Desoxyribonuclease and coagulase activity of micrococci. U. S. Armed Forces M. J., Tech. Bull., 9: , Elston, H. R., and Fitch, D. M.: Determination of potential pathogenicity of staphy- Am. J. Clin. Path, 32: , Esber, R. J., and Faulconer, R. J.: A medium for initial visual demonstration of production of coagulase and fermentation of mannitol bv pathogenic staphylococci. Am. J. Clin. Path., 32: , 1959! 10. Jeffries, C. D.: Comparison of six physiologic characteristics of staphylococci from laboratory specimens. Am. J. Clin. Path., SO: , Weld, J. T., Kean, B. H., and O'Leary, W. M.: Production of octadecenoic acid in plasma by Staphylococcus aureus. Proc. Soc. Exp. Biol. Med., 112: , Zebovitz, E., Evans, J. B., and Niven, C. F., Jr.: Tellurite-glycine agar: a selective plating medium for the quantitative detection of coagulase-positive staphylococci. J. Bact., 70: , 1955.