comparative evaluation five selective differential media ... · ing egg yolk. subsequently, egg...

APPLIED MICROBIOLOGY, March, 1965 Vol. 13. No. 2Copyright © 1965 American Society for Microbiology Printed in U.S.A.

Comparative Evaluation of Five Selective andDifferential Media for the Detection and

Enumeration of Coagulase-PositiveStaphylococci in Foods

F. D. CRISLEY, J. T. PEELER, AND R. ANGELOTTIMilk and Food Research, Robert A. Taft Sanitary Engineering Center, U.S. Public Health Service,

Cincinnati, Ohio

Received for publication 31 August, 1964

ABSTRACTCRISLEY, F. D. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), J. T.

PEELER, AND R. ANGELOTTI. Comparative evaluation of five selective and differentialmedia for the detection and enumeration of coagulase-positive staphylococci in foods.Appl. Microbiol. 13:140-156. 1965.-Five selective media for the detection and enumera-tion of coagulase-positive staphylococci were evaluated for their efficiency in therecovery of 17 strains of coagulase-positive staphylococci from foods. They were Staph-ylococcus Medium 110 (SM-110), tellurite-glycine-agar (TGA), egg-tellurite-glycine-pyruvate-agar (ETGPA), tellurite-egg-agar (TEA), and tellurite-polymyxin-eggyolk-agar (TPEY). Statistical analysis by the rank correlation method of the efficiencywith which these media recovered staphylococci from pure 24-hr Brain Heart Infusioncultures revealed the following efficiencies in descending order: (i) TPEY, (ii) ETGPA,(iii) TGA, (iv) TEA, (v) SM-110. Growth of 17 strains of coagulase-negative cocci onthese media showed the following approximate descending order of inhibition to theseorganisms: (i) ETGPA, (ii) TEA, (iii) SM-110, (iv) TGA, (v) TPEY. The appearance ofcolonies of the various coagulase-negative strains on each medium was studied for thedegree to which they could be confused with colonies of coagulase-positive strains.Nineteen food contaminants, including Proteus vulgaris, Bacillus sp., Escherichia coli,Erwinia sp., fecal streptococci, and others, were also studied for similarities in appear-ance to staphylococci and for ability to grow on the selective media. The influence of fivesterile food homogenates (frozen chicken and tuna pies, custard, smoked ham, and rawwhole egg) on recovery of 1,500 enterotoxigenic staphylococci (three strains) per milli-liter was determined by statistical analysis. Three main effects (culture, media, andfood) and three interactions (media with food, food with cultures, and media with cul-ture) were found to be significant. Recovery on TPEY was influenced less by food thanthe other selective media and showed optimal recovery ability from sterile custard,eggs, and ham. TGA recovered well from sterile chicken pie and custard, SM-110 fromsterile custard, and TEA from sterile ham. None of the media was outstanding in re-covering staphylococci from tuna pie. The ability of the five selective media to re-cover 1,500 enterotoxigenic staphylococci (three strains) per ml from three sterilefoods in the presence of 10 strains of contaminating bacteria added at the 0, 105, and106 levels per milliliter was also studied and analyzed statistically. Only three factorswere significant under these conditions-cultures, foods, and the interaction of mediawith the level of added contamination. Efficiency of recovery of TGA, SM-110, andETGPA was found not to be dependent upon the level of contamination. Recovery onTPEY decreased with increases in the number of contaminants. TEA increased in effi-ciency at the 105 level, but decreased at the 106 level. When recovery on Trypticase SoyAgar was considered to be 100%, the average percentage of recovery by each of theselective media under all experimental conditions was determined.

Many media have been proposed for the detec- toxigenic staphylococci presents special problemstion and enumeration of pathogenic staphylo- that limit the usefulness of methods designed forcocci from various habitats, based on character- clinical use. This has resulted in a continuingistics more or less correlated with pathogenicity. search for methods and media better suited forExamination of foodstuffs for potentially entero- use with food.

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MEDIA FOR COAGULASE-POSITIVE STAPHYLOCOCCI

Staphylococcus Medium 110 (SM-110) hasbeen widely employed for this purpose. It wasdesigned to take advantage of the ability of theorganism to grow in relatively high salt concen-trations and to enhance the properties of gelatinliquefaction (Stone reaction), mannitol fermenta-tion, and chromogenesis, all of which at varioustimes were thought to have been correlated withcoagulase production and pathogenicity. Of thesecriteria, chromogenesis is currently the mostwidely used by the food worker for selection ofstaphylococci on SM-110. Frequently, coagulase-positive strains from food either do not developthe characteristic pigment, or do so only afterextended incubation at room temperature orlower. This makes necessary the confirmation ofgreater numbers of isolates by the coagulase testand also delays the results, often beyond the timelimit of usefulness.The incorporation of tellurite into selective

media such as the tellurite glycine agar (TGA) ofZebovitz, Evans, and Niven (1955), a modifica-tion of the medium of Ludlam (1949), has beenvery useful. The striking black appearance ofstaphylococci on tellurite media after about 24 hreliminates the necessity of subjective judgmentsof pigmentation. Tellurite media are, however,widely considered to be inhibitory to manyfood-borne coagulase-positive staphylococci, andas such offer a distinct disadvantage in quantita-tive determinations of these bacteria. Moreover,Proteus vulgaris has been shown to grow out ontellurite glycine medium with the production ofblack colonies that closely resemble, and may beconfused with, those of coagulase-positive staphy-lococci (Deneke and Blobel, 1962; Crisley, un-published data).

Recently, Finegold and Sweeney (1961) de-scribed a polymyxin-containing medium useful inhospital work. This medium was not consideredby Baird-Parker (1962a) to be entirely satis-factory for examination of foods.

Gillespie and Alder (1952) observed that mostof the coagulase-positive strains they studiedproduced opacity when grown in media contain-ing egg yolk. Subsequently, egg yolk was in-corporated into media for examination of foods(Carter, 1960; Herman and Morelli, 1960).Clearing of egg yolk in a selective medium hasalso been employed as a basis for selection ofcoagulase-positive staphylococci from platings offood samples (Hopton, 1961).Egg yolk has also been combined with tellurite

in selective media on which both development ofopacity (Inmes, 1960) and egg yolk clearing(Baird-Parker, 1962a) have been used as criteriafor identification of staphylococci in foods. Wehave employed a tellurite-polymyxin-egg yolk-

agar (TPEY) for investigations of commercialfoods, foods implicated in outbreaks of foodpoisoning (Crisley, unpublished data), and, morerecently (Crisley, Angelotti, and Foter, 1964), ofsynthetic cream pie fillings and pies. On TPEY,both the development of opacity and clearing inegg yolk are employed to identify staphylococci.The relative efficacy of diagnostic media for

staphylococci in food has not been adequatelyreported, particularly in the case of the recentlydeveloped recipes, and from the standpoint ofquantitative recovery, which is an importantfactor in examinations concerned with develop-ment and use of microbiological standards. Anevaluation of the egg-tellurite-glycine-pyruvate-agar (ETGPA) of Baird-Parker (1962a) wascompiled by him (Baird-Parker, 1962b) from theresults of various laboratories. The results showedthe method to be quite successful, but recoveryresults and other pertinent material were notincluded in the report.The present work was done to provide useful

data concerning the efficacy of five selective anddifferential media for determinations of Staphylo-coccus aureus in foods. The media were SM-110,TGA of Zebovitz et al. (1955), the tellurite-egg-agar (TEA) of Innes (1960), ETGPA of Baird-Parker (1962a), and TPEY of Crisley et al.(1964).

MATERIALS AND METHODSMedia. Trypticase Soy Agar (TSA; BBL) was

employed as a nonselective recovery mediumagainst which recoveries on the five selective me-dia were compared. It was prepared according tothe manufacturer's directions. The dehydratedversions of SM-110 (Difco) and TGA (Difco) weresimilarly made. TEA was made up according to theauthor's instructions (Innes, 1960). Potassiumtellurite for both TGA and TEA was purchasedfrom Difco. ETGPA was made up according toBaird-Parker (1962a, personal communication), byemploying potassium tellurite (The British DrugHouses, Ltd., Laboratory Chemical Division,Poole, England), since he considered this brand ofchemical to be essential. Glycine and sodium pyru-vate for ETGPA were purchased from NutritionalBiochemicals Corp., Cleveland, Ohio. Preparationof TPEY has been previously described (Crisleyet al., 1964).

Selective media were prepared, poured intosterile petri dishes, allowed to dry for 24 hr at roomtemperature, and used immediately or stored for1 week in a refrigerator before use, except forETGPA, which was poured and used within 24hr as recommended (Baird-Parker, 1962a).

Cultures. Sources of the 17 coagulase-positivestaphylococci are listed in Table 1. The 17 coagu-lase-negative cocci employed were isolated fromfrozen ocean perch (1), frozen codfish (1), frozenhaddock (1), turkey salad from a food-poisoning

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CRISLEY, PEELER, AND ANGELOTTI

episode (3), turkey meat from another outbreak(1), delicatessen potato salad (1), raw milk (2),frozen chicken pie (1), ham implicated in foodpoisoning (1), fomites (food tray, refrigeratorshelf) at the scene of a food-poisoning outbreak(2), nasal swab from a food handler (1), and Colbycheese (2). Common food contaminants (Table 3)were all obtained from the collection of the FoodMicrobiology Section.

All cultures were maintained on a semisolidstock culture medium (Crisley et al., 1964) at roomtemperature. Before use, they were subculturedonce in the same medium in a water bath for 24 hrat 35 C and seeded into tubes of Brain Heart In-fusion (BHI Difco) broth. BHI cultures wereincubated in the same way for 24 hr 4 30 min be-fore use. At this time, sterile glass beads wereadded, and each tube was mixed on a VortexJunior Mixer (Scientific Industries, Inc., Queen'sVillage, N.Y.) for 10 sec. A homogenous suspensionof bacterial cells was obtained that was remark-ably uniform in viable count from one experimentto another and could be diluted for mixed-culturestudies with reasonable certainty of the numbersadded.

Coagulase-positive staphylococci used in stud-ies of recovery from sterilized foods in pure ormixed cultures were grown on TSA slopes in Pyrexbottles. Cell suspensions were prepared from thesebottle cultures in phosphate-buffered dilutionwater (American Public Health Association, 1960),as previously described (Crisley et al., 1964).Food homogenates. Single lots of commercial

brands of frozen chicken and tuna pies were pur-chased at a local chain grocery store and werestored in a deep freeze at -20 C until needed. Alarge smoked ham was similarly obtained andstored. Custard filling was made up in the labora-tory according to the recipe of Angelotti et al.(1959). Raw whole egg was made by sterilizingfresh shell eggs in HgCl2 solution as for prepara-tion of egg yolk for TPEY (Crisley et al., 1964),and aseptically removing and blending the egg for20 sec in a Waring Blendor at low speed to mini-mize foaming. Frozen foods were thawed, cut upinto small pieces, weighed into large-mouth, screw-cap refrigerator jars, and sterilized in an autoclaveat 121 C for 15 min. Food homogenates, except forraw egg, which was used undiluted, were made ina ratio of 1 part food to 7 parts of buffer by adding64 g of sterile food to 448 ml of standard buffereddilution water (American Public Health Associ-ation, 1960). The resulting 1:8 homogenates con-tained the greatest concentration of solid foodsthat could be pipetted with accuracy.

Recovery studies. Surface plating was employedby delivering 0.1 ml of dilutions of cultures orseeded food homogenates to the surface of tripli-cate plates of media and spreading the inoculaover the surface with sterile glass spreader bars.Seeded plates were incubated at 35 C and countedafter 24 and 48 hr on a Quebec colony counter,except for counts of mixed cultures in foods on

SM-110, which were incubated for 48 hr at 35 C

and additionally for 72 hr at room temperature toencourage optimal pigmentation.Recovery on sterile and experimentally contam-

inated foods was studied on food homogenatesinoculated with approximately 1,500 staphylococciper milliliter, so that 0.1 ml contained 150 orga-nisms when delivered and counted on each plate ofTSA control medium. It was felt that this level ofinoculum provided approximately the smallestnumber of organisms that would respond to theeffects studied in a manner consistent with sta-tistical validity. In addition, the placement of un-duly large quantities of homogenate on each platesurface was avoided.

In the studies of recovery of staphylococci fromexperimentally contaminated foods, approxi-mately equal numbers of each of 10 common foodcontaminants were added to the staphylococcalinoculum at two levels: 105 and 106 bacteria permilliliter of homogenate. This means that eachplate of selective medium received totals of 104 and105 contaminants, together with approximately150 staphylococci. The contaminants were fourcoagulase-negative cocci (one each isolated fromColby cheese, raw milk, frozen chicken pie, andham from a food-poisoning outbreak); P. vulgaris4669; Bacillus sp. No. 5; B. subtilis H.; Alcaligenesfaecalis; Escherichia coli, M. C. 34; and Strepto-coccus faecalis. These species of contaminants wereselected because of their demonstrated ability togrow on the media studied, or their commonpresence in food.

After incubation, the plates of selective mediacontaining contaminants in mixed culture withstaphylococci were counted for colonies presumedto be S. aureus by the criteria accepted for eachmedium. Confirmation on a limited scale was ac-complished by fishing characteristic colonies toTSA slants from plates at both levels of con-tamination after all incubation periods, incubat-ing the slants for 24 hr at 35 C, and testing forcoagulase by the tube method, with the use offresh frozen human plasma and a 4-hr incubationperiod at 37 C. Since the coagulase-negative cocciand P. vulgaris strains used in the mixed-culturestudy were those bacteria previously noted toresemble coagulase-positive staphylococci on thetellurite-containing media, it was felt that themethod constituted a fairly stringent test.

RESULTSRecovery of pure cultures of coagulase-positive

staphylococci. Comparative growth of the 17strains recovered from 24-hr BHI broth culturesis presented in Table 1. After 24 hr of incubationon TPEY, colonies were convex and about 1.0 to1.5 mm in diameter, they were jet-black ordark-gray in color, and they exhibited one ormore of three types of egg yolk reaction. One wasa discrete zone of egg yolk precipitate around andbeneath the colony. The second was a clear zoneor halo often occurring together with a zone ofegg yolk precipitation beneath the colony. A

142 APPL. MICROBIOL.


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third type of reaction was the absence of either aprecipitation zone or halo, but visible precipita-tion beneath the colony. All strains showed thesetypical characteristics, except 196E, C-33, andP-1, in which the egg yolk zone was slightly morediffuse ("fuzzy") in appearance, but all othercharacteristics were typical. Egg yolk reactionswere generally better after 48 than after 24 hr.The zones of clearing of egg yolk on ETGPA

were not as clear or definite in a number of strainsas might have been expected from the report ofBaird-Parker (1962a). This is presumably due tothe tendency of the medium to remain moretranslucent in our hands than is desirable, thoughwe attempted to follow closely the directions andprecautions of Baird-Parker (1962a, personal com-munication). Frequently, an oily zone, somewhatsimilar to a clear zone, was discernible, and this,together with the colonial morphology and clear-ing, was accepted as the criterion for coagulase-positive staphylococci on ETGPA. Thiirteenstrains showed the slight clearing reaction in 24to 48 hr. Clearing was accompanied by develop-ment of an egg yolk precipitate in five strains. Ac-cording to Baird-Parker (1962a), the precipitationis characteristic of coagulase-negative Staphylo-coccus saprophyticus or occasional strains of S.aureus after incubation for 48 hr.

Furthermore, the variation in colony size ofcoagulase-positive strains after both 24 and48 hr was troublesome on ETGPA and was muchmore marked than on the other media. Colonysize generally ranged from pinpoint to about 1.0or 1.5 mm in diameter. We feel that this variationmay lead to lower counts in some foods when theextremely small colonies are overlooked, becausethey are so similar to coagulase-negative colonies.On TEA, the appearance of coagulase-positive

colonies was much closer to the published descrip-tion (Innes, 1960) after 48 hr of incubation thanafter 24 hr. Colonies did not appear to take upsufficient tellurite for clear differentiation afteronly 24 hr. The clear or oily zones around colo-nies, described by Innes (1960), occurred in 11strains, and clearing was accompanied by, orfollowed by, egg yolk precipitation in nine strains.Inhibition or atypical reactions at 24 hr wereexhibited by four strains.Appearance of colonies of coagulase-positive

staphylococci on SM-110 and TGA conformed tothe accepted criteria for these media.

Table 1 presents the number of coloniesenumerated on the selective media, expressed asthe percentage of the counts observed on TSAagar, which were considered to be 100% forpurposes of this study.The relative efficiency of the media studied was

determined by a rank correlation method re-

ported by Kendall (1955). The media were rankedin Table 1 by rows (horizontally) for both the24- and 48-hr incubation periods; the rank of 1was assigned to the medium exhibiting the highestrecovery, and subsequent ranks were assigned inthe descending order of recovery. Identical scoreswere resolved by averaging the ranks; i.e., twomedia that possess identical scores but shouldoccupy ranks 4 and 5 would each be considered asranking 4.5 (yielding a sequence of 1, 2, 3, 4.5,4.5). The sum of ranks was compared for each ofthe five media vertically (down the column). Acoefficient of concordance was then calculatedand tested to determine whether significantconcordance existed according to Kendall's(1955) method.The null hypothesis that the five media did not

grow staphylococci in any set order from the 17cultures was proposed. The alternative hypothe-sis is that the media do grow staphylococci in aset order from the 17 cultures. It was decided tolet the null hypothesis be rejected one time in 20when it is true (a = 0.05). Since N (number ofmedia) was less than seven, the value of S (sumof squares of column rank totals) must be com-pared with a critical value in the tables for(number of cultures) and equals 17; N equals 5.The critical value for S is 421.0. For an S valuelarger than 421.0, the null hypothesis was re-jected (the fact that the ranks were unordered).By this method it was concluded for both the

24- and 48-hr data that the value of S exceeded421.0 (i.e., S24 hr = 1,790.5 and S48 hr = 1,804);hence, the media were considered to be ordered.The best estimate of the ranks of the media indescending order of efficiency of recovery ofcoagulase-positive staphylococci was 1-TPEY,2-ETGPA, 3-TGA, 4-TEA, and 5-SM-110.

Recovery of pure cultures of coagulase-negativecocci. Expressed as the number of strains re-covered at various percentage levels of the TSAcount, the relative recovery of coagulase-negativecocci is presented in Table 2. Better outgrowthoccurred after 48 hr of incubation than after24 hr. SM-110 seemed to be somewhat moreinhibitory to coagulase-negative cocci than TGA,particularly after 24 hr, when the appearance ofcolonies on TGA was distinctly different from theusual appearance of coagulase-positive strains,though colonies of seven of our strains on TGAstrongly resembled positive types after 48 hr.Our results again confirm the desirability of a24-hr incubation period for TGA as recom-mended by Zebovitz et al. (1955) and otherworkers, and also that many coagulase-negativeorganisms grow out in 48 hr on SM-110, necessi-tating further incubation for pigment develop-



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TABLE 2. Recovery of 17 strains of coagulase-negative cocci on five selective media and

Trypticase Soy Agar

No. of strains recovered atvarious percentage levels of

Time of the TSA count (100%)*Medium incuba- -

tion01

0.0 to 0.11 11 to 51 to over0.1% to 50% 100% 100%

hr

SM-110 24 14 0 0 3 048 5 2 3 6 1

TGA 24 9 4 1 3 048 4 5 4 3 1

TPEY 24 5 1 1 6 448 0 0 2 9 6

TEA 24 11 0 1 3 248 4 0 3 7 3

ETGPA 24 14 3 0 0 048 11 3 1 2 0

* TSA counts per milliliter for the 17 strainsranged from 250 X 105 to 65 X 106.

ment and coagulase tests on both pigmented andunpigmented colonies.Of the three egg yolk recipes tested, ETGPA

(Baird-Parker, 1962a) was most suppressive tocoagulase-negative cocci. None of the visiblecolonies growing out at 24 hr resembled coagulase-positive types. After 48 hr of incubation, how-ever, four strains produced colonies similar tocoagulase-positive strains, morphologically and inegg yolk-clearing ability, which Baird-Parker(1962a) considered to be a characteristic ofpositive types. One of these strains also showedprecipitated egg yolk beneath the colony.TEA (Innes, 1960) was less inhibitory to

coagulase-negative cocci than was ETGPA. Twoof our strains produced colonies that resembledcoagulase-positive strains according to the cri-teria of Innes (1960). Our turkey-meat straindiffered only in that the colony was slightly morebrown than the gray or black color usually asso-ciated with coagulase-positive colonies.Of the egg yolk media, TPEY was least inhibi-

tory to coagulase-negative cocci. After 24 hr,coagulase-negative cultures produced coloniesthat were usually small (pinpoint to 0.5 mm indiameter), and white, gray, or black, with a lightperiphery. Usually there was no discrete zone ofclearing or precipitation of egg yolk. In four ofour strains, on crowded areas of the plate, therewas occasionally a diffuse type of egg yolk pre-cipitation quite different from the discrete zonalreaction noted with coagulase-positive colonies.Some of our strains, after 48 hr of incubation onTPEY, had developed larger dark colonies similarto coagulase-positive organisms, but had not

developed an egg yolk zone, or had shown onlythe diffuse type of reaction. One strain, ourturkey-meat isolate, developed a zone of clearingand egg yolk precipitate similar to those as-sociated with coagulase-positive colonies. Thisstrain also gave trouble on ETGPA and TEA.

Growth of common food contaminants. Growthof 19 bacteria that may be encountered in food issummarized in Table 3. Generally, none of themedia was completely inhibitory to A. faecalis,members of the genus Bacillus, and P. vulgaris.Particular attention was paid to the appearanceof P. vulgaris colonies on the media as a result oftheir resemblance to staphylococci on tellurite-containing media (Deneke and Blobel, 1962;Baird-Parker, 1962b; Crisley, unpublished data).Outgrowth of P. vulgaris on TPEY was notcompletely suppressed, but was markedly lessthan on TGA or ETGPA. Only a few of thecolonies present were equal in size to staphylococ-cal colonies. They were flatter, more brown, andshowed no egg yolk reaction. P. vulgaris showeda tendency to spreading film formation onETGPA, which increased with the number ofsuccessive rapid transfers prior to plating. Al-though the organism grew out on TEA, there wasconsiderable inhibition, and the colony mor-phology was characteristically different fromstaphylococci. In several other cultures, acoalescing film of growth was present on platesof the 10-1 dilution of broth culture, but therewas no growth at higher dilutions.The Bacillus cultures that we tested grew

slightly better on TPEY and TEA than onETGPA, TGA, and SM-110. Bacillus growth onTEA was especially troublesome, because itshowed a tendency to spread more extensivelythan on TPEY. Bacillus sp. were easily differ-entiated from staphylococci.

Factorial analyses of (i) the effect of sterile foodsand its interactions with media and cultures and(ii) the effect of the level of experimental contami-nation and its interactions on the recovery ofcoagulase-positive staphylococci. Essentially thesame statistical models and assumptions weremade to determine the relative effect of theseaspects on recovery on the five media as related tooverall recovery on TSA. The analysis of theeffect of sterile food is based on data from Table4 and the effect of experimental contaminationon data from Table 5. Triplicate plates werecounted for each of the 75 combinations of fivefoods, five media, and three cultures, and theresults (225 observations in all) are presented inTable 4. In the analysis of the effect of experi-mental contamination (Table 5), triplicateplates were counted for all combinations of fourfactors (i.e., three cultures, five media, three

VOL. 13, 1965 145


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TABLE 4. Influence of five sterile foods on the recovery of Staphylococcus aureus 196E, LAB-i, and71 on five selective media and Trypticase Soy Agar in a typical experiment

Food homogenate

Frozen chickenpie

Frozen tuna pie

Laboratorycustard

Smoked ham

Concentratedwhole raw egg

Plate counts of triplicate plates after 48 hr of incubation at 35 C

SM-110 TGA TPEY TEA ETGPA TSA

196- LAB-E 1

198178146

159156174

184186179

127163128

161119121

129138137

169156150

164155131

1108197

7710075

71

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132114109

1109184

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618896

196-E

164177176

204161186

172138160

129137114

149156162

LAB-1

168161139

114111139

144155159

7410777

1079190

71

15799164

119108103

11510383

10910898

117119133

196-E

138145173

169176198

196139220

129126134

184163219

LAB-1

158167165

118152131

145148153

141147148

164133136

71

103111129

125118126

9896128

968896

1129486

196-E

129178143

162195158

125161136

144150116

127150139

LAB-1

968994

8710495

106107116

140150138

165130128

71

948396

90101141

766673

9192100

807481

196-E

105118123

151138162

10499101

130133132

181184207

LAB-1

145126136

139163144

95102102

8010186

13610399

71

97102113

115101103

967372

595462

877977

196-E

163176165

178208177

198201161

180105147

184173195

LAB-

157155165

133130121

132127140

142209113

87102118

foods, and three levels of contamination byconcomitant bacteria). The data used in Table5 were averages of triplicate plate counts in a

typical experiment (135 observations in all).The factors were tested for average differences

within the factors, such as the differences amongthe five media, among the three foods, etc., andthe first-order interactions among the food,media, cultures, etc. The mathematical modelsfor factorial experiments involving data fromTables 4 and 5 were as follows. Model for testingdata in Table 4:

Yiikl = ,u + ai + Pi + -Yk + Ti + (af3)i;+ (ay)ik + (fiY)ik + eijkl

i=1,*' ,5j= 1, *- 5k=1,- ,3I= 1, * 3

Model for testing data in Table 5:

Yiikl = A + ai + P + (a)Mj + -Yk + (aY)ik

+ (#3Y)jk + 8i + (ab)il + (#S),I + (-Y)kl + Eijkt

i=1,v~,3ij= 1, *93,k=1,-** ,3

I= 1,*-- ,3

where: Yijkl = the log plate count in the ijklthcell; , = overall log mean; a i = the ith food

effect; ffl = the jth media effect; Yk = the kth

culture effect; Tl = the Ith replicate effect;

TABLE 4a. Analysis of variance for factorialanalysis of culture, media, and

foods at 48 hr

Sum of Degrees MeanSource squares of free- square F-ratio

(SS) dm (MS)

Replications.. 0.00134 2 0.00067 0.15Media ........ 0.29291 4 0.07322 16.02*Food ......... 0.25289 4 0.06322 13.83*Culture ....... 1.30438 2 0.65219 142.71*Media Xfood ........ 0.38265 16 0.02391 5.23*

Media Xculture ..... 0.09662 8 0.01207 2.64*

Food Xculture. 0.11301 8 0.01412 3.09*

Error......... 0.82352 180 0.00457Total......... 3.26732 224

* Significant at a = 0.05.

51 = the Ith level of contamination effect;(a1)i; = the ijth media X food interaction;(a'y) ik = the ikth food X culture interaction;(ab) it = the ilth food X level of contaminationinteraction; (3'Y)jk = the jkth media X cultureinteraction; (flb)ji = the jlth media X level ofcontamination interaction; ('Yb)kl = the klthculture X level of contamination interaction;-ijkl = the ijklth error effect.

71

157118178

106122105

139838619374119

969284

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TABLE 4b. Media recovering significantly more Staphylococcus aureus for each food and culture*

Food homogenates Cultures

Frozen tuna Frozen chicken Custard Raw egg Ham 196E LAB-i C-71pie pie

None TGA TPEY TPEY TPEY TPEY TPEY TGATGA TEA TGASM-110 SM-110

* Where more than one medium is listed, they showed approximately equal trends that are signifi-cantly larger than the remaining media.

The assumptions chosen for the mathematicalexperiments were as follows. (i) The observationsYijkl are random variables distributed abouttrue means that are fixed constants. (ii) Themodel contains additive effects. (iii) The Yijklare independently distributed in a normal dis-tribution. (iv) The random variables Yijkl havea common variance, ac2. (v) The model used is afixed-effects model (i.e., we limit our conclusionsto the foods, cultures, media, and level of con-tamination actually present).

Auxiliary assumptions were that the platecounts made on the experimentally contaminatedplates were all S. aureus colonies (i.e., colonies ofother bacteria were not counted as S. aureus),and that SM-110 plates that were incubated for5 days to allow for pigment development wereassumed to behave like the other selective mediaat 48 hr of incubation. These assumptions weregenerally borne out by previous experiments,though confirmation by coagulase test was neces-sarily limited by the volume of work.The plan of statistical analysis (Ostle, 1954)

was to test the effects within the factors andbetween the factors of the first-order interactionsto determine whether any of them were differentfrom zero. All tests were performed on the as-sumption of rejection of the hypothesis that themain effects and the interaction effects areequal to 0 only once in 20 times when the hy-pothesis is true (a = 0.05).To assure that two of the assumptions in

Table 4b were satisfied, logs were taken to makethe count data approximately normally dis-tributed, and Bartlett's tests (Snedecor, 1956)were performed to examine homogeneity ofvariances. The Bartlett's test confirmed sta-tistically the experimental observations that the24-hr counts were frequently incomplete, owingto the erratic outgrowth of staphylococci andvariations in the diagnostic reactions. Hence,only the data collected after 48 hr of incubationat 35 C were analyzed.

Analyses of variance for factorial analysis ofthe main effects, first-order interactions, and the

experimental error are presented in Tables 4aand 5a for the experiments on sterile food ho-mogenates and the experimentally contaminatedsystems, respectively. Variation in techniquesmay include, among other things, a gain inefficiency, owing to experience in counting plates.

Other quantities in the tables include thesum of squares (SS), which are divided by thedegrees of freedom (df) to get the mean squareestimate (MS) for the main effects and the inter-actions. Ratios of mean squares to the meansquare error are listed in the F-ratio column. Ifthere is no effect due to the particular mainfactor or interaction being tested, the F-ratiowill be near 1. As the F-ratio becomes larger, itcan be said that there is more evidence thatthere is an effect due to the main effect or inter-action in question. For example, if a factor suchas the culture effect is found to be significant onthe average, some strains tend to grow out betterthan others. To test which strains were signifi-cantly different from each other, the test sug-gested by Duncan (1955) would be used. If aninteraction is present, some quantities of onefactor significantly influence the effect of an-other.

Effect of sterile foods. Based on the data inTable 4, the analysis of variance presented inTable 4a indicates that three main effects andthree interactions were significant. They wereculture, media, and food; and interactions mediax food, food X culture, and media X culture.By Duncan's (1955) test, the 196E strain wasrecovered in greater numbers than LAB-1, andthe LAB-1 strain was recovered in larger numbersthan the C-71 culture when logarithms of theplate counts were averaged over the whole ex-periment.On TPEY, recovery was better than on TGA

and SM-110; on TGA, it was better than on TEAand ETGPA. Moreover, recovery of staphylococciwas better from tuna and chicken than fromcustard, eggs, and ham.

Figures 1, 2, and 3 present the trends in theinteractions. In these figures, points close to the



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CRISLEY, PEELER, AND ANGELOTTI APPL. MICROBIOL.

TABLE 5a. Analysis of variance for factorial analysis of culture, media, foods, and level of contaminationat 48 hr

Source Sum of squares Degrees of Mean square (MS) F-ratio(SS) freedom (df) Ma qae(S -ai

Media ................................ 0.07963 4 0.01240 2.37Level of contamination ................... 0.02273 2 0.01136 2.17Food ................................ 0.16361 2 0.08180 15.64*Culture .................................. 0.47692 2 0.23846 45.59*Media X level of contamination........... 0.09393 8 0.01174 2.24*Media X food ............................ 0.07177 8 0.00897 1.71Media X culture .......................... 0.06233 8 0.00779 1.48Level of contamination X food . .......... 0.03689 4 0.00922 1.76Level of contamination X culture ......... 0.04147 4 0.01036 1.98Food X culture . ......................... 0.03797 4 0.00949 1.81Error ................................ 0.46105 88 0.00523Total ................................. 1.54830 134

* Significant at a = 0.05.

ordinate r = 0 show little or no interaction withthe quantities of the factors given on the abscissa,as compared with points lying to either side ofr = 0. Points above "0" indicate a positive inter-action as measured by an increase in numbers ofstaphylococci recovered. Points below "0" indi-cate negative interaction in terms of a decreasein staphylococcal recovery.For example, in Fig. 1, TPEY was influenced

less by food than were the other selective media,and SM-110 was greatly influenced by raw eggsin which the count was reduced. Marked positiveinteractions occurred between TGA and chickenpie; SM-110 and custard; TEA and ham; andbetween ETGPA and raw eggs and tuna pie.Negative interactions took place between TGAand ham; SM-110 and eggs; TEA and chicken;and between ETGPA and custard and ham.

Food-culture interactions (Fig. 2) were lessimportant than media-food interactions. Positiveinteractions occurred between 196E and raw

egg and between LAB-1 and custard. Negativeresults were found between C-71 and custardand 196-E and chicken.

Culture-media interactions (Fig. 3) had even

less effect than food-culture interactions. Positivevalues were found between LAB-1 and TPEYand between C-71 and TGA. Negative valueswere shown between LAB-1 and TGA.The question of which medium recovered the

most staphylococci when employed with a

particular food or culture was answered byemploying the statistical method of Scheffd(1953), which allows determination of the signifi-cance of certain comparisons after the data havebeen examined. Table 4b indicates the mediathat recover significantly more organisms foreach food and culture at the a = 0.05 level. Onthis basis, TPEY can be recommended for

cc

w

0

Z +0.144-

WZ

(DW

Ji +0.10

7iB

+0.060>

WT,0 +0 02

Ir

> -0.02r:0WZ

o <

-0.06

Jc-0.10w

Q.

0 TUNA* CHICKEN

CUSTARD

A EGGS* HAM

A

* 0 0

A 0

8 0 ~~~00

*

0

A

TPEY TGA SM-110MEDIA

TEA ETGPA

FIG. 1. Interaction effects between sterile food ho-mogenates and staphylococcal selective media after 48hr of incubation at 85 C, based on logarithms of in-dividual plate counts.

optimal recovery from custard, eggs, and ham;TGA from chicken pie and custard; SM-110from custard; and TEA from ham. None of themedia was outstanding in recovering staphylo-cocci from tuna pie.Three media (TPEY, TGA, and SM-110) were

outstanding in recovering 196E. TPEY alsorecovered more organisms of the LAB-1 strain,and TGA was best in recovering C-71.When recovery on TSA was considered to be

100%, the calculated average per cent recoveries

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VOL. 13, 1965 MEDIA FOR COAGULASE-POSITIVE STAPHYLOCOCCI

a0

0

LL

w0.-z

, o

w

2

0

09

>

Z ZZ

IAJ

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O0:00:v0-c-j

0.03

0.02

0.01

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-0.01 _

-0.02 _

-0.03 _-

AOzLABA.196E OD a C-71

0

o 0

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A~~~ A -

0

0

o

A 0

HOE C El- ~~TUNA CHI CKEN CUSTARD EGG HAM

FOOD

FIG. 2. Interaction effects between sterile foodhomogenates and test cultures after 48 hr of incuba-tion at 85 C, based on logarithms of individual platecounts.

UJ

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ti <0

LAJ +crw

zr

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wZ

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A = 196E

z 0= -71

0

0

OA

A 0

A

A

-0.01

0 0-0.02

-0.03 e-0

TPEY TGA SM-IIO TEA ETGPA

MEDIA

FIG. 3. Interaction effects between selective staphy-lococcal media and test cultures after 48 hr of incu-bation at 85 C, based on logarithms of individualplate counts.

of staphylococcal media under all conditionsin the experiment on sterile foods were: TPEY,99.3%; TGA, 93.0%; SM-110, 91.5%; TEA,83.0%; and ETGPA, 80.3%.

Effect of experimental contamination. Fac-torial analysis of main effects and interactionswas carried out on data presented in Table 5for the effect of three levels of experimentalcontamination (0, 105, and 106 contaminants).The estimate of absolute recovery on eachmedium as compared with recovery on TSA fromsterile food homogenates under identical condi-tions is included in the overall calculations.The analysis of variance (Table 5a) shows

that only two factors and one interaction reachedthe level of significance. They were cultures andfoods and the interaction media X level of con-tamination. Duncan's (1955) test was again usedto show differences between quantities withinfactors.

Significantly (a = 0.05 level), more colonieswere found on platings of 196E and of LAB-1and more on platings of LAB-1 than of C-71.The culture factor did not interact significantlyat the first-order level with the other factors;that is, better growth of one strain when com-pared with another seemed to be independent ofthe other factors.

Platings of chicken pie revealed significantlymore colonies than platings of custard, which inturn showed more colonies than platings of eggs.Averaged over the experiment, the three levels

of added experimental contamination (0, 106, and106 bacteria per milliliter) showed no significantdifference among media. Only one of the sixfirst-order interactions (media X level of con-tamination) was found to be significant at thea = 0.05 level.

Figure 4 shows the effect of the interactionbetween media and contamination level. Theratio in Fig. 4 should be "0" if the efficiency ofthe media is not dependent on the level of con-tamination. The media TGA, SM-110, andETGPA are nearly "0" for all three levels, butTPEY decreases in recovery with an increase inthe number of contaminants, and TEA peaksat a level of 100,000 added contaminants permilliliter. TPEY and TEA seem to be responsiblefor the largest part of the interaction of media Xcontamination level.When recovery on TSA was considered to be

100%, the calculated average per cent recoveriesof staphylococci in the presence of experimentalcontamination under all conditions was: TGA,97.2%; TPEY, 94.4%; TEA, 93.0%; SM-110,90.1%; and ETGPA, 83.8%.

151


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IO 0

A = IOOT

0- IM-

0

A

a_0

_ A A

A 0

A

0

0

SM-l 10 TGA TPEYMEDIA

TEA ETGPA

FIG. 4. Interaction effects between selective staphy-lococcal media and the level of experimental contami-nation after 48 hr of incubation at 35 C, based on

logarithms of the means of triplicate plate counts.

DISCUSSIONWe feel that the three egg yolk media generally

possess advantages for differentiation of co-

agulase-positive staphylococci over media with-out egg, in spite of conflicting opinions concerningthe value of the egg reaction as a diagnostic tool.Gillespie and Alder (1952) found 80% of coagu-lase-positive staphylococci from human sources

outside the hospital environment producingopacity in egg yolk media, whereas all of their 60coagulase-negative organisms were egg yolk-negative. Similar results were found.by Alder,Gillespie, and Herdan (1952). Based on clearingand precipitation of egg yolk, only 76 egg yolk-positive strains of 186 coagulase-positive S.aureus strains were isolated from umbilical stumpsof newborn infants by Graber et al. (1958). Thesame source yielded 14 egg yolk-positive strainsof 24 coagulase-negative strains. Nevertheless,Graber et al. (1958) considered the egg yolkreaction diagnostically helpful, because 95 strainsof S. albus, S. citreus, and S. aurantiacus were

egg yolk-negative on plates, and their pigmenta-tion is not always sufficiently distinct from S.aureus on other media. Silverman et al. (1961),working with isolates from shrimp, also feltthat the egg reactions increased their abilityto find staphylococci in their samples, sincecoagulase-positive staphylococci were not iso-

lated from samples that did not yield egg yolk-reacting colonies. Carantonis and Spink (1963)found that only 11 of 505 opacity-producingstrains (60% from lesions and the rest fromnasal swabs of health carriers), considered bythem to be "pathogenic," were coagulase-nega-tive. These authors also found 98 of 101 "patho-genic" egg yolk-negative strains to be coagulase-positive. However, 100 additional "presumptivelynonpathogenic" egg yolk-negative strains werealso found to be coagulase-negative. The resultsof Carantonis and Spink (1963) indicate a heavypreponderance of coagulase-positive culturesamong isolates from clinical sources showing apositive egg yolk reaction. In the study of variousfoods, the egg yolk reactions (either clearing oropacity) were found to be superior to othercriteria in differentiating staphylococci by Hopton(1961), Baird-Parker (1962a, b), Carter (1960),Innes (1960), and Jay (1963).On the other hand, in their studies of isolates

from hospital patients, Mandel and Warholak(1962) found 362 of 382 (95%) coagulase-positivestrains and 73 of 142 (51%) coagulase-negativestrains to produce clearing and opacity on eggyolk media. Although they felt that the fre-quency of positive egg yolk reactions in coagulase-negative strains (51%) nullified the differentialvalue of the egg yolk reaction, Mandel andWarholak (1962) noted that most of theircoagulase-negative, egg yolk-positive cultureswere from the nose, throat, and abscesses; and themajority of coagulase-negative, egg yolk-negativestrains were from urine cultures. This apparentsource specificity of the egg yolk reaction shouldbe further investigated. If the relationship werefound to be a firm one, it might prove to bevaluable in determining whether foods have beensignificantly contaminated in handling, sinceimportant sources of staphylococci in food arethe respiratory tract and lesions of food handlers.The presence of coagulase or egg yolk reactivity

may not always be a reliable indicator of entero-toxigenicity. Silverman et al. (1961) called at-tention to variations in both of these propertieswithin strains or substrains of staphylococci.Coagulase-negative strains derived from positivecultures have been noted. The present avail-ability of serological methods for enterotoxindetection (Casman, McCoy, and Brandly, 1963;Hall, Angelotti, and Lewis, 1963) greatly reducesthe need for animal tests. More accurate andcomprehensive work directed at re-examiningthe relationship between enterotoxin productionand coagulase (and other attributes of staphylo-cocci) than was heretofore possible should becarried out.The results clearly show that recovery of

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staphylococci from pure laboratory cultures isonly one of several factors that must be con-sidered in selecting a medium for analysis offood. For example, though TPEY was clearlysuperior in this respect to the other media testedand also in recovery from sterile foods, it re-covered more coagulase-negative cocci and lostsome of its recovery efficiency in experimentallycontaminated foods. We feel that the chiefvalue of the present work is to shed light onsome of the factors that should be considered inthe choice of a selective medium for the analysisof foods for staphylococci. In addition, severalrecommendations can be made.TPEY was designed by us for optimal recovery

as a result of many experiments on a few teststrains of staphylococci. Its relatively low levelof inhibitory effect on coagulase-negative cocciis probably directly related to its superior re-coverability of coagulase-positive strains. ETGPArecovered from pure cultures nearly as well asTPEY, but without allowing the coagulase-negative strains to grow. TGA was less inhibi-tory to coagulase-positive staphylococci thanwas SM-110. This may not be true of staphylo-cocci on primary isolations from food, becausemany workers have expressed contrary opinions.The studies on pure cultures indicate, to some

extent, the accuracy of diagnosis that can beexpected on the five media. The great variationin size of colonies on ETGPA in our hands mustbe considered a disadvantage to be numberedamong others already reported (Baird-Parker,1962a, b). Of the egg yolk recipes, TPEY showedfewer atypical results than the other egg media,though one of the coagulase-negative strainsshowed strong egg yolk reactions usually identi-fied with coagulase-positive types. This is notunexpected, in view of the reports in the litera-ture. On the egg yolk media, generally, a 48-hrincubation period yielded better results thanwere obtained at 24 hr with our laboratory teststrains. We have, however, noticed that onactual platings of food samples the egg yolkreaction is often more pronounced after 24 hrthan in our test cultures. Although we count andselect colonies on food plates on the basis of adiscrete egg yolk zone, consideration must begiven to the production of diffuse egg yolk re-action zones by some coagulase-positive cul-tures on TPEY, since three of our strains showedtendencies to produce diffused egg zones. Gen-erally, our experience with foods shows thatwhere such zones appear in the colonies incrowded areas of the plate, they are indicative ofcoagulase-negative cocci. When they appear inwell-isolated colonies, we make a practice of

coagulase testing a few to make certain of theirreaction.With a few exceptions, approximately the

same species of miscellaneous contaminantstended to grow out on all of the selective media.This may simply reflect the possession by themedia of common mechanisms for selection ofstaphylococci from food. The two strains of P.vulgaris studied were generally less troublesomeon TPEY than on ETGPA or TGA. We feelthat only minimal experience with TPEY wouldbe necessary to distinguish easily these Proteusstrains from staphylococci. Even more trouble-some than its morphology on ETGPA was thespreading tendency of P. vulgaris, which mightresult in overgrowth of staphylococci on Baird-Parker's medium.

Baird-Parker (1962b) pointed out that P.vulgaris also grows out on the polymyxin agar ofFinegold and Sweeney (1961) and the egg yolkazide agar of Hopton (1961), as well as on TGAand ETGPA. He attempted to overcome thisdeficiency in ETGPA by the addition of phenyl-alanine and detection of phenylpyruvic acidproduced by P. vulgaris on this substrate.The appearance of coalescing films of growth

with several other common food contaminantsat the 10-l dilution, but absence of any growthat higher dilutions, is suggestive of the presenceof metabolic products in high enough concentra-tion to overcome the suppressive effects of se-lective media. A similar situation is possible inplatings of heavily contaminated spoiled foods.Growth of Bacillus sp. on the selective media

is important, because members of this genushave been shown to be inhibitory to staphylococci(Graves and Frazier, 1963). Production of anti-biotics by members of the Bacillus group is wellknown. TEA particularly suffered from Bacilluscontamination, but TPEY was also considerablyaffected.

Analysis of the effect of sterile food confirmsstatistically the frequent observation that cul-tural differences among strains are of primaryimportance. They were the most importantsingle factor in our experiments. Differencesamong media and foods were second and third,respectively, in rank of importance.

Paralleling its performance in recovering purecultures from BHI broth, TPEY recovered bestfrom sterile foods and appeared to be markedlybetter than the other two egg yolk media.ETGPA, in contrast to its ability to recover purecultures, did not recover staphylococci from foodas well as the other selective media, a goodexample of the discrepancy that may occur be-tween recovery of pure cultures and recoveryfrom food. Higher overall recovery on all media

153VOL. 13, 1965


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from tuna and chicken pies than from custard,eggs, and ham may be an indication of thepresence of an inhibitory substance in the latterthree foods or the presence -of a "clumping"factor, such as has been reported to occur infresh raw milk (Smith, 1957). This point, how-ever, requires further testing.Our analysis of the main effects and significant

interactions in the sterile food experiments mayalso help to explain some of the presently con-flicting data and opinions regarding the efficacyof various selective media for food work. Mediashowing positive values (above 0 in the figures)would be most valuable in quality control workor other situations requiring relatively sensitivestaphylococcal determinations. These media arelisted in Table 4b, which is based on the test ofScheffe (1953). For example, TGA would appearto be best for analysis of chicken pie and TPEYfor raw egg. For overall general use, TPEY mightwell be the medium of choice, since it appears torecover well with three of the five foods. On theother hand, if custard samples are presentlybeing run on SM-110, no benefit will accruefrom a change to TPEY, especially if laboratorypersonnel are experienced in the use of the oldermedium.To a large extent, it is possible to make such

recommendations from our experiments, be-cause under our conditions culture X food andculture X media interactions were less signifi-cant than those of media X food. In othersystems, it is quite possible that different inter-actions would assume greater significance. Hence,more studies of a variety of foods and mediamight tend to broaden and solidify recommenda-tions for standardized methods that could profit-ably be utilized for better quality control offoods. Whether such studies are of sufficientvalue to be undertaken at all is dependent onthe scientific and economic importance of theresults. Development of meaningful microbio-logical standards in foods, for example, wouldseem to require such studies.The effect of experimental contamination

tended to alter the relative plating efficiency ofthe media noted in the experiments on sterilefoods. As main effects, cultures and foods werenot significantly changed from results in sterilefoods. The interaction between media and levelof contamination illustrated in Fig. 4 clearlyshows that recovery on two media, TPEY andTEA, was affected. The reason for the apparentincrease in counting efficiency on TEA in thepresence of approximately 100,000 added con-taminants is not clear and requires further testing.A very definite "experience factor," which maybe partly responsible, was, however, noted in

our study-that is, counting efficiency tended toincrease on all media in proportion to our ex-perience with them. Nevertheless, in the presenceof comparatively large numbers of experimentalcontaminants, TPEY, the medium that we foundleast inhibitory to coagulase-positive staphylo-cocci, was reduced in efficiency, and this must beconsidered if this medium is to be used in qualitycontrol and other work involving large numbersof similar contaminants. TEA also suffered fromthe same deficiency, apparently because of thepresence of Bacillus organisms, which seemed tospread extensively on this medium.

Because no significant culture X media orfood X media interactions were found to existin the face of experimental contamination, thecultures and foods were essentially constanteffects for the five media-that is, the five mediarecovered essentially the same numbers of thethree cultures relative to each other in the con-tamination experiment. If present results werealso true for the large number of cultures en-countered in routine food work, then a specificmedium-culture effect does not have to be con-sidered. While it may be convenient for theroutine worker to accept the absence of a mediumX culture effect (because it presently cannot bepredicted for all staphylococcal strains in exist-ence), it is necessary for the researcher concernedwith evaluation of media employing a fixed groupof strains, to consider this effect. The same re-lationship may also be true of other contami-nated foods, even though a food-media inter-action may be present in sterile foods, as we havealready shown.Our results indicate that TPEY is generally

superior in recovering staphylococci from purecultures and foods, though other media may beapproximately as useful for specific purposes.Its efficiency in the presence of contaminants maybe expected to vary directly with the number ofcontaminants. To a great extent, the loss inefficiency of recovery would also necessarilydepend on the type of contaminants present inthe food, a variable that cannot be tested ex-perimentally beforehand. Furthermore, our ob-servations suggest that the worker's experiencewith a selective medium cannot be overlookedin judging the efficacy of such media when themany variables are considered.Our experience with TPEY thus far indicates

that the egg yolk-tellurite reactions as employedin this medium are highly satisfactory for analysisof a variety of foods, despite the small percentageof unusual coagulase-negative types that maygive a characteristic egg yolk reaction. Of 91isolates (from market foods and foods involvedin food-poisoning outbreaks) considered pre-



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MEDIA FOR COAGULASE--POSITIVE STAPHYLOCOCCI

sumptively coagulase-positive on the basis of theirmorphology and egg yolk reactions, 84, or 92%,were confirmed by the tube test. In one instance,we isolated and identified by the egg yolk re-action two staphylococcal colonies on a platecovered with an almost solid film of growth ofother food contaminants. To date we have notnoticed an adverse effect on staphylococcal re-covery from market and outbreak foods ofheavy contamination by concomitant bacteria,including Bacillus species. This may be partiallydue to what appears to be more pronounced eggyolk reactivity by food isolates on primary isola-tion as well as experience with our medium. Forthese reasons, its superiority in recovering staphy-lococci, and the fact that recovery on TPEYappears to be less influenced by food constituentsin sterile foods than the other media tested, wefeel that the extra work and care necessary inpreparing TPEY should generally yield betterresults in the examination of a wide variety offoods for staphylococci. A more definite recom-mendation must await the accumulation offurther data and experience by us and others.Our experience also indicates that poured platesof TPEY can be stored in a refrigerator for ap-proximately 1 week before any change in se-lectivity is noted. After this time, the mediumseemed to become less inhibitory to our coagulase-negative staphylococcal strains.

ACKNOWLEDGMENTSWe sincerely appreciate the excellent technical

assistance of Nancy Batterson and Gayle Mul-berry.

LITERATURE CITED

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