Comparison of Direct Plating Media for the Isolation and Enumera-tion of Enterococci in Certain Frozen Foods1
MARY K. BtTRKWALL AND PAUL A. HARTMIAN
Department of Bacteriology, Iowa State University, Ames, Iowa
Received for publication 23 August 1963
ABSTRACT
BURKWALL, _MARY K. (Iowa State University, Ames), AND
PAUL A. HARTMAN. Comparison of direct plating media for theisolation and enumeration of enterococci in certain frozen foods.Appl. MIicrobiol. 12:18-23. 1964.-A total of 15 agar mediawere examined for their yield, selectivity, readability, andsimplicity of preparation and use. A thallium medium of Barnes
was selected as the better of the high yield-fair selectivity typeof medium and an azide-citrate medium of Reinbold appearedto be the better of the low Yield-high selectivity type of medium.Sodium carbonate (optimal concentration, 0.20%Z,) was found toincrease recovery substantially when added to certain media,especially in the presence of 0.05% Tween 80. When these twoingredients were incorporated into a medium modified afterSlanetz and Bartley, the resultant medium was superior toother media for the isolation and enumeration of enterococci incertain frozen foods, such as peas and hamburger, by the directplating method.
One of the first selective media for the isolation ofstreptococci was described by Fleming (1932), who usedtellurite to inhibit the growth of gram-negative bacteria.The same year, Edwards (1932) published a report on theisolation of "mastitis streptococci" from mixed cultureswith sodium azide as a selective agent. Later MIcKenzie(1941) proposed the use of thallium acetate as a third typeof agent for the selective isolation of streptococci. Othermaterials that have been used to impart selectivity toculture media for the isolation of streptococci includecrystal violet (MIcKenzie, 1941), penicillin (White andSherman, 1944), trypan blue (Chapman, 1946), ethyl violet(Litsky, MIallmann, and Fifield, 1953), sodium citrate(Reinbold, Swern, and Hussong, 1953), tetrazolium salts(Reinbold et al., 1953; Barnes, 1956), esculin (Seelemannand Obiger, 1956), and acridine orange (Raibaud et al.,1961). During the past three decades, a large number ofmedia have been formulated for the isolation and enumer-
ation of enterococci by most probable number, pour plate,and membrane filter techniques.Although many media have been in use, there have been
few comparative studies to determine which medium (ormedia) would be most suitable for a specific purpose. Inonly a few instances have more than two types of media
I Journal Paper No. J-4690 of the Iowa Agricultural and HomeEconomics Experimiient Station, Ames, IoWa. Project N). 1379.
for enterococcal isolation been compared simultaneously(e.g., Fanelli and Ayres, 1959; Splittstoesser, Wright, andHucker, 1961). The only recent comparative study ofmedia by use of the pour plate method is the work ofSaraswat, Clark, and Reinbold (1963) on dairy products.Since the enterococci are believed to be of value for thedetermination of fecal contamination of food, especiallyfor frozen foods (Hartman, 1960; Wilkerson, Ayres, andKraft, 1961) and dried foods (Kjellander and Nygren,1962), and since the pour plate method is quite practicalfor routine laboratory use, the present study was under-taken.
MIATERIALS AND MIETHODS
Agar media that have been used for isolation of strepto-cocci are listed in Table 1. MIedia examined in the presentstudy are denoted with an asterisk. Some media were notexamined because of the similarity in constitution to mediathat were studied (Table 1). The composition and pro-cedure for use were as recommended by the respectiveauthors or manufacturer, with the following exceptions.The pour plate method was utilized solely for all media;thallium acetate was substituted for thallium sulfate, andferric ammonium citrate was added to replace the ferricion supplied by blood which was excluded from medium16; and glucose was added prior to autoclaving ofmedium 6.
Samiiples of frozeii peas, green beans, corn, and hami-burger, obtainied froni a local retail store, served as theexperiiimental miiaterial. Commercial samples, rather thanexperimilentally inoculated foods or laboratory cultures,were used, because it was believed important to examiiinethe relative efficacy of the media under practical condi-tions. Although this mlethod of evaluation of media did notyield as definitive results and although difficulties were ex-perienced in assessinig the selectivity of the media, it wasfelt that these disadvanitages were miiore than offset byusing maaterial in which the physiological state of the bac-teria was the samiie as that occurring naturally in theproduct. The samples were kept frozen at all times, sincethawinig and refreezing resulted in significantly decreasedbacterial counts.
Samples for bacteriological analysis were removed byuse of a metal spatula, which was previously sterilized by
dipping into alcohol and then flaming. Plating procedureswere made according to Hartman and Huntsberger(1960). Of each sample, 50 g were weighed into a tared1-liter Waring Blendor container. The sample was homoge-nized at high speed in 450 ml of sterile 0.1 % peptone-water. Vegetables were blended for 2 min, and meatproducts were blended for 3 min. Two dilutions of eachsample were plated in duplicate on each enterococcusmedium. Two replicate portions of each sample were
plated, so that an estimate could be made of within-sample variation.
Isolation procedures were as follows. (i) Approximately25 colonies per medium per sample were selected andstreaked onto Trypticase Soy Agar (BBL). (ii) Approxi-mately 20 colonies from the Trypticase Soy Agar streakplates were inoculated into 0.25-oz dropping bottlescontaining about 10 ml of sterile Brain Heart Infusion(BHI; Difco) broth. (iii) When the culture had grown toform a turbid suspension, the organism was inoculatedinto each test medium in tubes (13 by 120 mm). Eachtube contained about 5 ml of medium and was coveredwith an aluminum cap. When an inoculum of two dropswas used, typical enterococci could be identified within24 hr.
Criteria for the identification of the enterococci were
those given by Sherman (1937); growth at 10 C, 45 C,and pH 9.6; growth in the presence of 6.5% NaCl and 0.1 %methylene blue; and survival at 60 C for 30 min. Typicalenterococci had to fulfill all six criteria. Those that were
negative in one characteristic, usually growth in 6.5%NaCl broth, were considered atypical. Those that fulfilledfewer than five of the criteria were assumed to be typesother than enterococci.
Various methods were examined for possible use infurther classification of the isolates. Carbohydrate fer-mentation tests, reaction with group D sera, and tetra-zolium reduction tests were of little use in identificationof the cultures. Tellurite reduction seemed to be of con-
siderable value for the differentiation of Streptococcusfaecalis and its varieties (which reduce tellurite strongly)from S. faecium (Orla-Jensen, 1919) and S. durans (whichreduce tellurite weakly or not at all). (See also Papavas-siliou, 1962.)Four primary criteria were used in the evaluation of the
different direct plating media: yield or comparativecounts; selectivity of the medium; readability, i.e., theease with which the colonies could be counted; and ease
or simplicity of preparation and use of the medium. Thislatter factor would be extremely important if the mediumor media of choice were to be utilized routinely. The
TABLE 1. Selective agar media for isolation of streptococci
(0.0015%)21 Reinbold et al. (1953)* NaN3 (0.01%), sodium citrate (2%), tetrazolium blue (0.001%)22 Slanetz and Bartley (1957)* NaN3 (0.04%), TTC (0.01%)23 White and Sherman (1944) NaN3 (0.03%), penicillin (3.25 IU/ml)24 Winter and Sandholzer (1946) NaN3 (0.04%), methylene blue (0.001%), penicillin (6.5 IU/ml)* Denotes media examined in the present study.t Incubation temperature 37 C unless stated otherwise.
objective of these studies was to discover which mediumor media would selectively affect a high recovery of entero-cocci from frozen foods containing various nonenterococcaltypes.
RESULTS AND DISCUSSION
Differentiation of colonies from food fragments in thefirst five media of Table 1 was facilitated by use of thetetrazolium plate flooding technique of Solberg and Proctor(1960). Although the readability of these media was
improved, these five media were excluded from laterstudies because of low recovery of enterococci comparedwith other media, or because they were not sufficientlyselective (see Table 2). Medium 12 (Table 1) was alsoeliminated from further consideration because of poorreadability and low recovery; this medium containedbromothymol blue, and most enterococci formed greencolonies that were difficult to differentiate from fragmentsof green peas and green beans. The blood in medium 19 alsointerfered with enumeration of colonies, and the selec-tivity of this medium for enterococci was poor; therefore,this medium was considered to be inadequate for thepresent purposes. Media 6 and 10 were the most satis-factory of the media discussed in this paragraph; however,a 24-hr incubation period was frequently insufficient forformation of colonies of a size adequate for enumerationin medium 6. In 48 hr, many pinpoint, red colonies notonly made enumeration on medium 6 quite difficult, butresulted in reduced selectivity as well. In spite of thisshortcoming, medium 6 was selected for further exami-nation because the yields were usually substantiallygreater than on medium 10.The esculin azide media (9 and 17) were found to give
relatively low recoveries of enterococci, but were quiteselective, usually supporting growth only of S. faecalisand its varieties. These two media, however, were rathercomplicated to prepare. Results obtained on a thallium-
TABLE 2. Comparison of recovery and per cent enterococcal isolationupon 14 agar media
Mediumn* Range of per cent recovery in Range of per centrelation to Barnes' medium enterococci
crystal violet-esculin medium (16) were similar to thoseobtained on the two azide esculin media (9 and 17),although many modifications were made with the formermedium (16) to adapt it for use with the pour plate method(Table 1). It was also noted that not all colonies werepositive for hydrolysis of esculin within the requiredincubation period, and, if both colorless (esculin-negative)and black (esculin-positive) colonies were counted, non-enterococcal types were included in the counts obtained.This occurred rarely on the esculin azide media (9 and 17)and frequently on the other esculin medium (16). Medium11 also fell into the group of low recovery, high selectivitymedia, but its readability was very poor compared withmedium 21. Medium 21 yielded counts and possessedselectivity similar to those obtained on the esculin andethyl violet media; however, preparation of the formermedium (21) was much easier, and readability of positivecolonies was much better. Therefore, medium 21 appearedto possess the most desirable properties of the mediadiscussed in this paragraph.Attempts were made to inierease the selectivity of
medium 6 and to modify medium 21 so that greaterrecovery would be obtained. Medium 6 was modified byvarying the concentration of thallous acetate (0.1 to 1.5 %),replacing the 1% glucose with 0.4% citrate, varying theconcentration of tetrazolium (0.01 to 0.1%), and incu-bating at 45 C. Medium 21 was modified by decreasingthe concentration of citrate (from 2 to 1.2%). All modi-fications resulted in no improvement in the desiredproperties of the media, and another approach to theproblem was undertaken.
Chesbro and Evans (1959) studied some factors af-fecting the growth of enterococci in highly alkaline media.These authors found that 0.05 M (0.53 %) Na2CO3 and0.05 % Tween 80 facilitated isolation of S. faecium fromfecal samples in Tryptone-yeast-glucose broth (pH 10.0).Based on their work, preliminary experiments wereundertaken with Tween 80 and Na2CO3 in media 6, 21,and 22. (The latter medium had not been examined inprevious experiments because of its similarity to certainother media tested.) There was a definite stimulation ofgrowth (increase in colony size and count) when frozenvegetable samples were plated on medium 22 containingfreshly prepared Na2CO3. Large, easily readable red col-onies were usually formed after 24 hr, and always after48 hr of incubation.When the concentrated Na2CO3 solution was auto-
claved [as specified by Chesbro and Evans (1959)], aloss of carbonate was apparent, as stimulation was greatlyreduced when a 4-day-old autoclaved solution was used.There also were great differences in the effect of Na2CO3in the various media used. At high concentrations ofcarbonate (approximately 0.5 %), a large amount ofprecipitate formed in medium 6. This precipitate didnot occur at a lower carbonate concentration (0.06 %-approximately the amount used as a bufer by Kenner,
Clark, and Kabler, 1961) which gave some growth stimu-lation. High concentrations of carbonate appeared to beinhibitory to recovery on medium 21, whereas lowerconcentrations were possibly somewhat stimulatory.The cause of growth inhibition and precipitate formationin medium 6 was believed to be the high pH (above pH10) formed at higher carbonate levels.Medium 22 was selected for further studies because
changes in pH caused by addition of different levels ofcarbonate were minimal. The original formula (Slanetzand Bartley, 1957) was modified by use of KH2PO4 inplace of K2HPO4 to lower the pH of the basal mediumprior to carbonate addition. Tween 80 (0.05%) was alsoadded. Concentrations of Na2CO3 varying from 0 to 0.5 %were incorporated into the medium prior to use. To pre-pare the 10% Na2CO3 solution, 10 g of Na2CO3 wereadded to 100 ml of sterile distilled water. This solutionwas not autoclaved, and control plates were made toinsure that there were no enterococci present in the solu-tion to increase plate counts from samples. The greateststimulation by carbonate was found to be at a level of0.20% (Fig. 1). Little or no loss in stimulation by carbo-nate occurred when the stock solution was stored for8 days prior to use. Increases in the counts on thesecond trial were believed to be due to higher microbial
180 - x
160 _ \ O FRESH 10% No2CO3I
SOLUTIONa. \ X - 8 DAY OLD 10%
, 140 _ \ No2CO3 SOLUTION
4wa-z 120-N0
Otoit
wa- 80
z
60wI-
a. 40-z4w2 20
0 .10 .20 .30 .40 .50% Na2CO3
FIG. 1. Effect of sodium carbonate concentration on enterococcalcounts obtained on a pea sample plated in modified Slanetz and
Bartley (1957) medium containing Tween 80.
populations in the outer portions of the frozen food massas compared with the microbial population in the center.This was found to be the tendency in most frozen peasamples. The drop in the recovery curve was not believedto be due to an increase in alkalinity (from pH 7 to 8),because later studies with NaHCO3 in place of Na2CO3yielded a similar curve. This would indicate that theincreases in count were not due to higher pH, sincedifferences in the pH of media containing the levels ofbicarbonate tested were negligible. Plates of medium 22were incubated in a CO2 incubator (20% CO2) and candlejar (approximately 10% CO2). Melted agar mediumsparged with CO2 prior to pouring the plates was alsoused. No increase in recovery was apparent with any ofthese treatments, which might suggest that a factorother than CO2 stimulation was responsible for increasedrecovery in the presence of Na2CO3. The cause of thedecreases in count at higher carbonate levels also is un-known. Identification of 104 isolates showed that atypicalenterococci [i.e., bacteria that fulfilled all of the Sherman(1937) criteria, except that growth in 6.5% NaCl wasscant after an incubation period of 7 days] contributedlargely to the increased counts obtained on the Tween-carbonate medium.
Curves with similar optima (Fig. 2) were obtained withand without the addition of Tween 80, when hamburgersamples were plated on the Tween-carbonate mediumcontaining various concentrations of Na2CO3. The countsobtained, however, were greatly increased with meatsamples when Tween 80 was present in the medium.Previous work [and that of Chesbro and Evans (1959)]indicated that recovery of S. faecium is enhanced by thepresence of Tween 80. Therefore, it is recommended thatTween 80 be added to media in which samples known orsuspected to contain S. faeciuin are to be plated.
Preparation and use of the Tween-carbonate medium,modified after the medium of Slanetz and Bartley (1957),was as follows: Trypticase (BBL), 15 g; Phytone (BBL),5 g; yeast extract, 5 g; glucose, 2 g; monopotassiumphosphate, 4 g; Tween 80 (Ruger Chemical Co., NewYork, N.Y.), 0.5 ml; agar, 15 g; and water, 1 liter. Theingredients were suspended in the water, and the mixturewas heated to boiling to melt the agar. This basal mediumwas dispensed (98 ml per bottle), and sterilized in anautoclave for 15 min at 121 C. For use, the agar wasmelted and cooled to 48 C, and the following additionswere made to each bottle of basal medium: 0.4 ml of 10%NaN3 solution (previously sterilized by steaming for30 min), 1 ml of 1 % 2,3,5-triphenyl-2H-tetrazoliumchloride solution [purchased in sterile form (from BBL)or prepared in the laboratory (with materials from East-man Organic Chemicals, Rochester, N.Y.) and sterilizedby steaming for 30 min], and 2 ml of 10% Na2CO3 orNaHCO3 (not sterilized). [Autoclaving the NaN3 in themedium was shown by Mieth (1960) to increase the in-hibitory properties of a constant level of azide. Because
of this factor, and the possibility that azi(media may become even more inhibitory uazide was not added to the basal medium uately prior to use.]
This medium was believed to be superior21, and 22 for enumeration of enterococci irsamples. Colony size was larger on the Twemedium than on any of the other media stu4ease of enumeration of colonies is a very impin its favor. Furthermore, yields of enterococto or better than those obtained on mediuiample, the per cent enterococci (based orcounts made on Trypticase Soy Agar plates i3 days at 30 C) from a cut green bean sam0.2, and 3.2%, respectively, for media 6,Tween-carbonate medium. Greater diffeobserved when other samples were plated.the Tween-carbonate medium were identificocci according to the criteria outlined in AMethods.Although three different types of sample
beans, green peas, and hamburger) were ulpresent studies, other samples and typesmay not yield analogous results. In additi
1200
w
-Ja.
4
(n
crw
c-
I
z
w
aI
-J
z
0 1 ' ' '0 .10 .20 .30
% Na2CO3
FIG. 2. Effect of sodium carbonate concentrationcounts obtained on a hamburger sample plated inand Bartley (1957) mediuim with and withoiut added
de-containingLpon storage,intil immedi-
to media 6,a frozen food,en-carbonatedied, and theortant aspectci were equalm 6. For ex-i total plateincubated forLple were 2.1,21, and the
medium is dictated, in large part, by the selectivity(groups of organisms) that one wishes to enumerate.The criteria utilized in the present studies to define se-lectivity will not be applicable under all conditions of use.However, it is believed that carbonate and Tween 80are helpful adjuncts to media for the isolation of atypicalenterococci, as well as improving the isolation of S. faeciumtypes.
ACKNOWLEDGMENT
This investigation was supported in part by U.S. PublicHealth Service grant EF-112 from the Division of En-vironmental Engineering and Food Protection.
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