Evaluation of Microbiological Methods Used for the Examination

of Precooked Frozen Foods12

HELEN ZABOROWSKI, D. A. HUBER, AND M. M. RAYMAN

Quartermaster Food and Container Institute for the Armed Forces, Chicago, Illinois

Received for publication August 15, 1957

The increasingly wide acceptance of precooked frozenfoods has led to the establishment throughout theUnited States of new frozen food plants devoted en-tirely to the precooked products. Among the problemsfacing this rapidly expanding industry is the develop-ment of adequate quality control standards for flavor,attractiveness, stability, and wholesomeness. As earlyas 1947 the question of wholesomeness was raised byFitzgerald, who pointed out the importance of highquality raw materials, adequate cooking, careful plantsanitation during processing and packaging, rapidhandling, and quick-freezing. Food sanitarians andpublic health officials presently are focusing their at-tention on the potential health hazards resulting fronunsanitary operations and improper handling durinigthe production and distribution of these foods. Foreffective sanitary control in the industry, processorsmay find it desirable to employ a standardized bacterio-logical testing program as an adjunct to rigidly super-vised measures of cleanliness. The purpose of this reportis to describe and evaluate laboratory methods used forestimating levels of bacterial populations surviving inthe precooked frozen products.When frozen meals were introduced into the U. S.

Air Force in-flight feeding program in 1951, a militaryspecification for these foods was published by theU. S. Army Quartermaster Corps. The sanitary require-ments specified for their production included a provisionfor continuous plant inispection by the Veterinary Corpsand imposed bacterial standards for the finished prod-uct. At present the specifieation (MIL-M-13966) pro-vides for a standard plate count not to exceed 100,000organisms per g, and a coliform plate count not toexceed 10 per gram. The two counts serve as an indexof ade(uate cooking. Since the relatively heat-labilecolon-aerogenes group of organisms cannot survive the

1 This paper reports research undertaken by the Quarter-rnaster Food and Container Institute for the Armed Forcesand has been assignied No. 631 in the series of papers approvedfor publication. The views or conclusions conitained in thisreport are those of the authors. They are not, to be construedas necessarily reflecting the views or indorsement of theDepartment of Defense.

2 Presented at the 56th General Meeting of the Society ofAmerican Bacteriologists held in HIouston, Texas, April29 to May 3, 1956.

time-temperature conditions of normal cookery, thecoliform count has also been a useful indicator of post-cooking contamination. The basis for establishingbacterial standards for precooked frozen foods pur-chased by the military was discussed in a preliminaryreport (Rayman et al., 1955) and will be dealt withmore extensively in a fortheoming publication (Huberet al., 1958). From those studies over a 5-year period,it became apparent that a need existed for specifyingreliable, simple, and rapid methods of analysis suitablefor control laboratories writh limited facilities.

For determination of standard plate counts thetentative methods for the microbiological examinationof frozen foods published in 1946 by the AmericanPublic Health Association, Committee on Microbio-logical Examination of Foods (APHA, 1946), provedto be relatively satisfactory. However, in some controllaboratories where frozen foods are subjected to bac-teriological testing it has beeni observed that a varietyof procedures differing from the APHA methods havebeen employed. The precision and accuracy of some ofthese 1nonstandard modifications have not been deter-mined and, particularly for specification purposes, thedata so obtained do not permit ready comparisons be-tweeni laboratories. Therefore, studies were made todetermiine wvhether the various details involved in thepreparation of food samples for analysis, such as weightof sample and method of dispersion, would influencethe bacterial counits. 1'reviously mentioned tentativemethods for frozen foods recommend an incubationtemperature of 32 C for 4 days and current APHAmethods for the examnination of dairy products permita choice of incubation temperature at 35 or 32 C for48 hr. Since minimal incubation times are generallydesired, comparisons of plate counts as influenced bytimes and temperatures were made. In addition to thetotal plate count and coliform plate count, the suita-bility of tests for enterococci and coagulase-positivestaphylococci was also inlvestigated.

Inadequate refrigeration often exists during traris-portation or storage of frozeni precooked foods. Ifthawing should occur with ensuing bacterial multipli-cation, and the food refrozen, the consumer may notbe warned of the potentially hazardous situation,although experts may recognize some evidence of

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H. ZABOROWSKI, D. A. HUBER, AND M. M. RAYMAN

refreezing. Since the heat treatment given precookedfrozen foods by the consumer is sometimes a mere oven-warming which cannot be depended on for destructionof organisms, the presence of even small numbers ofpotential pathogens in these items poses a problem forregulatory agencies. Tests for the coagulase-positivestaphylococci and for enterococci are warranted whenthere is reason to suspect that precooked frozen foodshave been subjected to inadequate processing or tomishandling as described above. Simpler, more rapidmethods for isolating and identifying these organismsare needed.

Preliminary surveys in this laboratory indicate thatcoagulase-positive strains of staphylococci are not in-frequently found in precooked frozen foods. Evidencethat such strains are pathogenic, enterotoxin-producingtypes has been reported by Evans and co-workers(1950). Many methods proposed for detection of theseorganisms rely on selective media containing high con-centrations of sodium chloride, thereby permitting thedevelopment of staphylococci while suppressing thegrowth of most other bacteria (Hill and White, 1929;Chapman, 1945). Liquid media of choice, reported byAMaitland and Martyn (1948), containing 10 per centadded salt, proved highly satisfactory for purposes ofenrichment. Zebovitz et al. (1955) devised a telluriteglycine agar which was particularly selective for iso-lating coagulase-positive staphylococci. Utilizing theselective properties of both sodium chloride and tel-lurite, the present report describes a modified methodsuitable for detecting small numbers of these micro-cocci in frozen foods.The role of enterococci as food-borne pathogens has

been a subject of much debate (Moore, 1955). In recentyears bacteriologists have shown considerable interestin the use of enterococci as an index of fecal pollutionin water, and some (Larkin et al., 1955) have proposedits substitution in lieu of the long established coliformindex. Burton (1949) suggested that enterococci whichsurvive for long periods of time in fresh frozen foodsmay be a better index of fecal contamination than thecoliform group which decreases in number during proc-essing and storage.

In 1937 Hartman found that the use of sodiumazide suppressed the growth of gram niegative bacteriawhile permitting the streptococci to grow. Since thisdiscovery, several investigators (Hajna and Perry,1943; Winter and Sandholzer, 1946; Rothe, 1948; iINall-mann and Seligmann, 1950; and Hajna, 1951) have de-vised selective media for isolating enterococci. Thesewere tested for suitability in the analysis of precookedfrozen foods. Although they are highly selective forenterococci, it was found desirable to use a confirma-tory medium such as the ethyl violet azide broth

[VOL. 6

MIATERIALS AND MIETHODSThe majority of foods used in this study were pre-

cooked frozen tray and casserole meals produced for

U. S. Air Force contracts (Military MIeals). In addition,

commercial precooked frozen meals, pot pies, and

2j1 pound bulk pack fresh frozen vegetables ere pur-

chased either from frozen food distributors or retail

supermarkets. Wherever possible, all test samples in a

set were from the same lot. Generally, a minimum of

5 meals was analyzed from a lot which usually consistedof a day's production of one menu (1000 to 3000 meals).Sample preparation. Each component of a precooked

frozen meal was analyzed as a separate entity. The

entire (85 to 170 g) frozen block of meat (or potato or

other vegetable) was chopped into approximately 1-in.pieces with a sterile knife. The frozen pieces were

weighed either in a sterile wide-mouth glass-stopperedbottle or a tared blender jar. A 1:5 suspension was

made by adding one-half of the measured volume ofsterile distilled water to the pieces in the blender jar,blending for 1 min, adding the remaining diluent andblending for an additional 2 min. The 1: 10 dilution wasmade by pipetting 50 ml of the 1:5 suspension into a

sterile, 50-ml buffered water dilution blank containingglass beads. Additional dilutions were made in bufferedwater as required. Military casserole meals were

stripped aseptically of their baked crusts and 100-gsamples chopped from wedge-shaped sections of thefillings. These 100-g amounts were then prepared as

described above. The unbaked top crusts of a fewvcommercial pot pies (chicken and beef) were analyzedseparately in the prescribed manner. However, for themajority of the commercial samples the crusts were

discarded. In the analysis of spaghetti and meat ballcasseroles, 50 g of meat plus 50 g of spaghetti were used.When samples blended in a Waring Blendor were

compared with those shaken in bottles with diluent andglass beads, a 1:5 initial suspension was prepared foreach method. Samples of 50 g were blended in theblender jars with 200 ml of sterile distilled water, and

25-g samples were shaken in bottles with 100 ml ofsterile distilled water and a teaspoon of glass beads.

Standard plate count. For each appropriate dilution,1 ml portions were inoculated into duplicate platesusing milk-protein hydrolysate glucose agar (BBL) as

the plating medium. Unless otherwise stated, incubationwas at 32 C for 72 hr. For experiments comparing 32and 35 C incubation, two sets of duplicate plates were

prepared, one set incubated at each temperature. Whenincubation times were compared, plates were countedafter 48 and 72 hr.

Coliform plate count. Coliform counts were made induplicate on serial dilutions prepared as describedabove, in accordance with Standard Mlethods for theExamination of Dairy Products (APHA, 1953), usinig

(Litsky et al., 1955) for verification.

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8MICROBIOLOGICAL EXAMIINATION OF IPRECOOKED FRtOZEN FOODS

Detection of coagutlase-positive staphylococci. Tuibes oftrypticase soy broth (BBL) containing 10 per cenitadded NaCl were inoculated in duplicate with 1 mlamounts of the 1:5 and 1: 10 suspensions of sample andincubated for 48 hr at 32 C. From these enrichmentcultures, phenol red mannitol agar (Difco) containing10 per cent salt (PRMS-10 per cent) and/or telluriteglycine (TG) agar3 plates were streaked and held 24 hrat 37 C. Typical staphylococcus colonies were pickedto Bacto-brain heart infusion broth and incubated at37 C. After 18 to 20 hr, cultures were stained for verifi-cation as staphylococci, and coagulase tests were made.Directions for the test are provided with ampules ofBacto-coagulase plasma. When TG agar was usedalone, the staphylococcus cultures from brain heartinfusion broth were streaked on phenol red mannitolagar to demonstrate mannitol fermentation.

Detection of enterococci. The following broth media(Difco) were selected for comparison: S F, enterococcuspresumptive (EP), azide dextrose (AD) and B A G G.For qualitative studies, duplicate tubes of each testmedium were inoculated with 1 ml quantities of the1:10 dilution. Incubation at 45 C for 3 days was usedfor all except azide dextrose broth which was incubatedat 37 C for 48 hr. Growth in the azide dextrose and acidproduction in the other three media was indicative of apresumptive positive test. Confirmation as enterococciwas accomplished by subculture in ethyl violet azide(EVA) broth. Two drops from each presumptive posi-tive tube were inoculated into EVA broth and incu-bated at 37 C for 48 hr. The presence of enterococci wasdemonstrated by turbidity and formation of a purple"button" at the bottom of the tube. Examination ofsmears made from the positive EVA broth culturesinvariably confirmed the presence of streptococci. Inpreliminary trials, cultures from more than 100 positiveEVA tubes were studied, employing the following con-firmatory tests for enterococci described by Sherman(1937): (1) growth at 10 and 45 C, (2) growth in brothcontaining 6.5 per cent NaCl, and (3) survival insterile milk after heat treatment at 63 C for 30 min.Since the presence of enterococci was continually re-

affirmed by these tests in the early stages of this investi-gation, a positive EVA test was subsequently acceptedas sufficient confirmation.

Quantitative studies were also employed for a com-

parison of azide dextrose broth with EP broth usingthe most probable number technique. In these studies,EP broth was preheated at 45 C before inoculation.Serial decimal dilutions (10-' to 10-r) were prepared,and for each dilution 3 replicate tubes were inoculatedwith 1 ml portions. M1ost probable numbers (MIP'N),

3Dehydrated tellurite glycine agar is now commerciallyavailable from Difco, Baltimore Biological arid Case Labora-tories. Initially, the medium was prepared as described by

based on confirmed positive tests, were computedaccording to the McGrady probability tables, (Bu-chanan and Fulmer, 1928).

RESULTS

Standard Plate Count

Methods of sample preparation. Cominercial samplesof precooked frozen beef pot pies, precooked frozenMexican corn (whole kernel corn combined with pi-miento and green pepper) and of fresh frozen cauliflower,green peas, and lima beans were analyzed by the blend-ing and shaking methods described previously. Resultsare given in table 1. The bacterial counts on the blendedsamples were higher, as was expected, in the majorityof the samples. One shaken sample of MIexican corn,

however, gave a much higher count. Each set of fivesamples was drawn from a single lot. This may havebeen due to the unequal distribution of corn, pimiento,and pepper; however, an attempt was made to chooseequal proportions.

Time of incubation: 48 versus 72 hr. Standard platecounts were made on 114 components of precookedfrozen meals (19 meats and 95 vegetables), to compare

the effect of 48- and 72-hr incubation periods. Themajority of the counts on the vegetables (92 per cent)

TABLE 1

Comparison of bacterial counts in frozen foods aising twomiethods of sample preparation

Standard Plate Count, Thousands per g

Product

Beef pot pieShaken ...............

Blended.Mlexican corn

Shaken ...............

Blended ..............

PeasShaken ...............

Blended ..............

Lima beansShaken ...............

Blended ..............

CauiliflowerShaken ...............

Blended ..............

Sample no.

3236

3960

4.816.5

3564

0.40.6

2

3765

5868

5.911.5

38110

0.7

1.0

5

120172

23055

1516

58110

0.72.6

3

5163

6566

815

38100

0.70.5

4

6654

66100

8.816.0

4699

0.71.8

TABLE 2

Comnparison of standard plate counts in precooked frozenmeats and vegetables incubated at 32 and 35 C

Percentage of Samples Having Counts

No. of SamplesComponent Examined Higher at Lower at Same at

32 C 32 C 32 and 35 C

Veget able 49 16 25 59

Meat 45 31 16 53

Zebovitz et al. (1955).

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H. ZABOROWSKI, D. A. HUBER, AND M. M. RAYMAN

were not influenced by the additional 24 hr of incuba-tion. On the other hand, 11, or 60 per cent, of the meatsamples gave higher counts after 72 hr.

Temperature of incubation: 32 versus 35 C. As shownin table 2, results from a total of 94 samples of meatsanid vegetables indicated that, in general, there waslittle difference between standard plate counts incu-bated at 32 or 35 C for 72 hr. A "difference" refers arbi-trarily to an average colony count difference of 5 ormore.

Isolation of Coagulase-Posilive Staphylococci

A comparison between TG agar and PRMS-10 percent agar wvas made, using parallel inoculations fromthe enrichment broth cultures. Of 93 frozen foodsamples examined in this manner, coagulase-positivestaphylococci were recovered from 32 samples on TGagaIr and from only 23 samples on PRMS-10 per centagar. All samples from which staphylococci were re-covered on PRMS-10 per cent agar were also foundpositive on TG agar. When enrichment broth cultureswere streaked on PRMS-10 per cent agar plates, anovergrowth of sporeforming rods occasionally maskedthe staphylococcus colonies, making it difficult toisolate them for further study. Streaking on TG agarprovided more effective inhibition of the sporeformers,permitting the easier isolation of staphylococci.

Comparison of Mledia for the Detection of Enterococci

Qu.alitative and quantitative tests for the detectionaind eniumeratioin of enterococci were made employingfour li(uid media: namely, S F, B A G G, EP, and ADbroths. In one series of qualitative tests for enterococci,131 samples were examined using S F broth alone. Of56 samples which wvere found presumptive positive, 3failed to givre a positive confirmatory test. Conductinga seconid series of qualitative tests, 100 samples wereexamined comparing B A G G broth with El' broth.Of these, 89 were positive in B A G G broth with nofalse positives, while 92 were positive in the E1' brothwith one false positive.

In the three selective media, S F, B A G G and EPbroths, the presence of enterococci is indicated by thefornmationi of acid. However, both the S F and 1B A G Gbroths which contain phosphate buffer and bromeresolpurple (BCP) indicator often failed to produce a clear-cut acid color change. Omi the other hand, satisfactorilysharp color changes were regularly observed with theEl' mediuim which omits the phosphate buffer land con-

tains bromthymol blue (BTB) indicator. Therefore, twomodifications of the B A G G broth formula were

tested, one substituting BTB indicator for the BC1',the seconid using the same indicator, BTB3, without thephosphate buffer. The sharpness of the color changewas not improved in either modification.

In a third series, quantitative methods were em-ployed comparing EP broth with AD broth. A total of57 samples representing 10 commercial lots of frozeiifoods were examined for most probable numbers ofenterococci using both media. EP medium recoveredenterococci from 55 items and AD broth indicated thatall samples were presumptive positive. EVA confirma-tion of these presumptive positive samples was ob-tained for 52 with the EP broth and 56 with the ADmedium. When the sum of the presumptive positivetubes in the three significant dilutions was considered,the 57 samples furnished a total of 229 cultures fromEP medium with 170 confirmations (74.2 per cent),whereas the corresponding values for AD broth were301 presumptive positive cultures with 209 confirma-tions (69.4 per cent).

Tvpical data, illustrated in table 3, show reliabilityof presumptive positive tests in the two media. It willbe noted that the number of presumptive positive tubeswN-as generally greater in the case of the AD medium.

Reliability of

Product

Chicken pot pie

Beef pot pie

Rloast turkey anddressing

Green peast

Parslev butteredpot a to

TABLE 3presumptive positive tests for enterococci in

precooked frozen foods

Enterococcus Pre-sumptive Broth

No.* r)re- No. con-sumptive firmedpositive positive

6 36 36 56 66 4

53136

64453

35554

54465

0

111

4

644

32

33554

53344

Azide Dextrose Broth

No. pre-sumptivepositive

76786

56766

a

6675

66689

86477

* Suim of positive tuibes in 3 signiificant dilutions.t Fresh frozein.

No. con-firmed

positive

64653

42334

55665

54335

56343

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MICROBIOLOGICAL EXAMINATION OF PRECOOKED FROZEN FOODS

The per cent of false reactions appears to vary some-what from lot to lot, and this range miay be gauged fromthe table.

In table 4 are showni estinmates of enterococcus pop-ulations in a few commercially frozen foods as obtainedby the two media. As noted before, results obtainedwith AD broth are generally higher than those withEP. It will be seen that vegetables such as green peas,which are uncooked, may be expected to have highenterococcus counts despite the fact that these productsare usually subjected to a mild blanch prior to freezing.Precooked frozen foods, on the other hand, possesslower enterococcus counts dependent on the extent ofcooking and the degree of recontamination duringpackaging.

DIscussIONOwing to the heterogeneous character of precooked

frozen foods, the bacterial content is likely to beunevenly distributed. Thus, Logan et al. (1951) found,for chicken d la king, that the bacterial content of the

TABLE 4Estimation of enterocci* in precooked frozen foods.

A comparison of two media

Most Probable Number per gProduct

Enterococcus Azide dextrosepresumptive broth broth

Chicken pot pie 25 15011 4075 1500150 7545 250

Beef pot pie 4 04 94 154 1545 20

Roast turkey and dressing 250 9575 9545 25025 25025 1100

Green peast 1100 750250 2500250 450045 2500

450 4500

Parsley buttered potato 25 25045 9520 95

450 2525 25

* Confirmed.t Fresh frozen.

gravy was quite low, whereas the chicken meat con-tained 70 per cent of the total count. If, in sampling,the proportions of chicken meat to gravy were notmaintained approximately equivalent to those of theprocessed product, the analytical results were subjectto appreciable error. When these foods are preparedfor analysis it is important to mininmize sampling errorsby weighing a relatively large sample and preparing auniform suspension so that the bacterial compositionof any small subsample is representative of the averagecomposition of the whole product. Therefore, in pre-paring samples for analysis, each component of a mealwas taken in its entirety and blended. A meal com-ponent, as referred to here, is an individual serving of ameat preparation or potato or other vegetable item.Such portions range in weight from approximately 3ounces (85 g) to 6 ounces (170 g), depending on thefood. Since many of the meat items weighed as much as6 ounces, the blender method was found most practicalfor obtaining uniform suspensions using the entiresample.As described above, a comparison was made of the

bacterial counts obtained from samples prepared byblending and by shaking with glass beads. The presentfindings confirm observations by Jones and Ferguson(1951) that use of the blender method results in slightlyhigher counts. Coarse suspensions of shaken materialsoften furnish mechanical difficulties in transferringsample aliquots and lead to pipetting errors. Conven-tional shaking with glass beads is insufficient to reducethe particle size of some precooked frozen foods and,despite the use of 50-ml volumetric pipettes withcut-off tips, plugging by food particles cannot beavoided. Not only is it easier to pipette the blendedsample, but it is also less time-consuming and thefrozen pieces of food are not subjected to a mere surfacewashing as they are in the shaking method. It will benoted that the practice of blending a frozen product inthe manner we have described does not permit thetemperature of the resulting suspension to rise above18 C, which is well below the incubation temperaturerange. Any heat generated during such a 3-min blendingis insignificant as it relates to possible heat destructionof bacteria in the sample.To further minimize sampling errors within produc-

tion lots, it is important that a sufficient number ofindividual packages be selected for analysis to representthe average bacterial content throughout the lot.Variations in counts which occur during the processingand packaging of precooked frozen foods are dictatedby the degree of sanitary control in the different plants,and this, in turn, influences the number of units neededfor a representative analysis of a lot. Wherever possiblein this study, at least five complete meals from a singlelot were examined with the objective of obtaining fairly

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11. ZABOROWSKI, D. A. HUBER, AND M. AM. RAYMAN

good representation. The present military specificationprovides for a minimum of three meals from eachday's production of 5000 units or less and one additionalmeal for each additional 2000 units per lot.

Incubation factors which influence the standardplate count appear to depend partly on the nature ofthe food and its accompanying microflora. Thus suchfoods as meat, containing large numbers of organismsproducing pin-point colonies, showed increased bac-terial counts when incubated longer than 48 hr. How-ever, counts for vegetables remained substantially thesame after 48 or 72 hr. For this reason it appears un-necessary to incubate plates longer than 3 days. Desic-catiOn of agar and interference from spreading coloniesare also thus minimized. The 72-hr incubation periodallowing pin-point colonies to develop enough to bemore easily differentiated from the background debrismay account for the higher bacterial count found in60 per cent of these meat samples. Most vegetables, onthe other hand, contained large sporeforming rods thatproduced large, easily recognized colonies within 48 hr.Bacterial counts of meats and vegetables were liot sig-nificantly influenced by varying the incubation tem-perature between 32 and 35 C.

Since the number of coagulase-positive staphylococcipresent in precooked frozen foods is usually very low inproportion to the total number of organisms, directstreakinig of low dilutions on a selective medium suchas PRAIS agar and TG agar was not satisfactory.However, if an enrichment medium such as trypticasesoy broth with 10 per cent NaCl added is employed, theisolation of these organisms becomes easier. In prelimi-i)ary studies, the inoculated enrichment broth that hadbeen inicubated at 32 C for 48 hr was streaked ontoPRAIS-10 per cent agar plates. Because cocci, otherthan the coagulase-positive staphylococci, and grampositive sporeforming rods also grew well on theseplates and produced acid, colony differentiation alonecould not be relied upon. When TG agar was used,gram positive rods were easily differentiated from thestaphylococci on these plates. The colonies producedby sporeformers were small and colorless or grey incontrast to the larger, entirely black, buttery coloniesof the coagulase-positive staphylococci. The latter wereconfirmed by staining and by the coagulase test. Con-firming the observations of Zebovitz et al. (1955) it wasfouind inadvisable to hold the TG plates much longerthan 24 hr because of the delayed appearance of coagu-lase-negative staphylococci which also produce blackcolonies. Since colonies of coagulase-negative staphylo-cocci occasionally appear within 24 hr, a test for coagu-lase production is essential.

Of the several media employed for the isolation ofenterococci, EP broth gave the most clear-cut results.The chanige from blue to bright yellow on acid produc-tion made the reading of a presumptive positive testmuch easier. Although AD broth seemed to be a more

[VOL. 6

sensitive medium, as seen by the greater number of con-firmed positive tests, the larger number of subculturesthat had to be made was more time-consuming. SinceEP broth gives good recovery of enterococci and thenumber of false positive reactions are few, it can beused satisfactorily on a qualitative basis for controlwork. Azide dextrose broth, on the other hand, is moreapplicable where greater accuracy for the quantitativeestimation of enterococci is needed.

It is desirable that the EP broth be preheated at 45 Cbefore inoculation to prevent the growth of strepto-cocci other than those of Group D, since growth at45 C is one of the differentiating characteristics of theenterococci. Although at 45 C this medium is highlyselective for enterococci, confirmatory tests are re-quired.

In his studies, Burton (1949) found that, as indicatorsof contamination, the coliform organisms appeared to besuperior to the enterococci in unstored or briefly storedfrozen foods, while the enterococci were a more reliableindex in foods which have been held in freezer storage.The majority of the military meals used for the presentanalytical studies had been stored about 4 months;however, the storage history of the commercial foodswas not always known. Coliform organisms in thestored precooked frozen foods should have been absentin most cases, since they are easily destroyed by heat.The results of these studies substantiate Burton's ob-servations. Coliform organisms were recovered fromvery few of the precooked frozen items except thosewhere the original bacterial count was relatively high,whereas enteiococci were found much more frequently.Presence of coliform organisms would indicate eitherthat the foods had been cooked insufficiently or thatthey had been recontaminated later in the processing.Presence of enterococci would also indicate under-cooking and possible recontamination during handling.A comparison of the recovery of coliform organisms

and enterococci is shown in table 5, where results ofanalyses on 414 samples wrere evaluated. These samplesincluded military and commercial precooked frozenmeats, vegetables, and pot pies, as well as commercialfresh frozen v-egetables. From a total of 234 itemsprepared under military specifications, coliform organ-isms were found only in 2.5 per cent of the samples, andenterococei in 41 per cent of the samples. Of 180 com-

mercially prepared items, 42.7 per cent not only con-

tained coliform organisms, but they were present inmuch greater numbers than in the military meals.Enterococci were present in 92.7 per cent of the com-

mercial samples. In the 42 samples of commercial freshfrozen v-egetables the coliform count was less than 10per g in 16.6 per cent; however, 78.5 per cent were

positive for enterococci.That coliform organisms and enterococci had been

found less frequently in precooked frozen foods pro-duced under military specifications may imply the need

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in commercial production for better cooking standards,better plant sanitation, and better quality of raw

materials. These data point up the fact that, when con-

tinuous supervision and inspection are exercised, a highquality standard for precooked frozen foods can be at-tained. Qualitative tests described above for detectingthe presence of staphylococci and enterococci are usefulaids in maintaining quality control. Further work isplanned to investigate quantitative population levelsof these organisms.

Incorporating the optimal features of the presentinvestigation, the authors suggest the following pre-

scribed procedure for the microbiological examinationof precooked frozen foods:

Sampling. Take 1 package from each of 5 shippingcases of the same production lot in question. (Adequacyof this sampling will vary in relation to the size of thelot and, if there is any significant variability in the lot,this fact will become evident in the majority of cases

through individual tests on the 5 samples.) Keepsamples frozen with Dry Ice (solid C02) or othersuitable refrigerant if analysis is to be delayed or

sampling point is at some distance from the laboratory.Preparation of sample. (a) Casserole meals-including

pot pies. Remove aluminum foil cover. Using asepticprecautions, with a sterile knife cut or chop wedge-shaped sections of the frozen meal (including the crust,if present) into approximately 1-in. blocks. Weigh 100 g

into a tared, sterile, screw-cap aluminum Waring (orsimilar type) Blendor jar.4 Measure into a sterile,graduated cylinder a volume of sterile distilled waternecessary to make a 1:5 suspension. Add asepticallyto the chopped material in the blender jar approxi-mately } to 12 the required amount of water. Blend1 min. Add the remaining water and blend for 2 addi-tional min. Prepare a 1:10 suspension by pipetting50 ml of the 1:5 suspension into a sterile, 50-ml bufferedwater dilution blank containing a teaspoonful of glassbeads. (To facilitate this transfer, the tip of a 50-mlvolumetric pipette should be cut off before sterilizationto provide a wide bore.) Shake the diluted samplerapidly at least 50 times through an arc of 1 ft in orderto insure homogeneity. Prepare consecutive decimaldilutions at the 1:100, 1:1000, and higher levels (ifnecessary) using sterile buffered water blanks.

(b) Multi-compartment tray meals. The contents ofeach compartment of a tray meal are analyzed as sep-arate entities. (Thus, for example, a frozen block con-

sisting of meat, gravy and dressing is considered an

individual sample.) Remove sufficient aluminum foil

cover to expose the compartment desired for analysis.Using aseptic precautions, cut or chop the entire frozen

4For convenience, samples may be weighed a day in ad-

vance, kept in freezer, and blended on the following day. In

this procedure, the chopped pieces of food are weighed directlyinto a tared, sterile wide-mouth bottle, placed in the freezer,and transferred aseptically to the blender jar at time of anal-

ysis .

block of the product (50 to 175 g) into approximately1-in. blocks with a sterile knife. Transfer all thesepieces aseptically to a tared, sterile screw-cap blenderjar and proceed as directed above for casserole meals.

Methods of analysis. (a) Standard plate count. Pre-pare duplicate plates from each dilution using 1 ml ofinoculum per plate. Mix thoroughly with about 15 mlof melted milk-protein hydrolysate glucose agar andincubate at 32 to 35 C for 72 hr.

(b) Coliform plate count. Prepare duplicate platesfrom appropriate dilutions using 1 ml of inoculum perplate. Mix thoroughly with melted desoxycholate lac-tose agar and incubate at 37 C for 18 to 24 hr.

(c) Coagulase-positive staphylococci. Inoculate du-plicate tubes of trypticase soy broth containing 10 percent NaCl with 1 ml amounts of appropriate dilutionof sample. Incubate at 32 C for 48 hr and from eachtube of the trypticase soy salt broth streak a loopfulonto a tellurite glycine agar plate. After incubation at37 C for 24 hr, pick typical black colonies (glossy and"buttery", 1 to 2 mm diam) to brain heart infusionbroth. Incubate at 37 C for 18 to 20 hr. Prepare smearsfrom these tubes to verify staphylococcus morphologyand test for coagulase. (Directions for test are providedwith ampules of the desiccated coagulase plasma.)

(d) Enterococci, qualitative tests. Inoculate duplicatetubes of azide glucose broth (10 ml per tube) with 1 mlamounts of the appropriate dilution of sample, andincubate at 37 C for 48 hr. A positive presumptive test,indicated by visible growth, requires confirmation forenterococci by subculturing 0.1 ml into ethyl violetazide broth, with incubation at 37 C for 48 hr. A con-firmed test is demonstrated by turbidity and the forma-tion of a purple "button" at the bottom of the tube.As an alternate medium for azide glucose broth,

enterococcus presumptive broth may be employed. Thismedium should be preheated to 45 C before inoculationand incubated at this same temperature. Although apositive presumptive test, indicated by acid production,

TABLE 5Recovery of coliform organisms and enterococci from military

and commercial precooked and fresh frozen foods

No. Samples No. Positive No. Positive forExamined for Coliform* Enterococci*

Sample

Cmeria Military Com M'Co-Militarymercia1Ml itary mercial tary merical

Precooked frozen 26 95 4 2 22 47meats

Precooked frozen 30 129 12 4 30 49vegetables

Chicken pot pie 45 10 30 0 45 0Beef pot pie 37 0 24 0 37 0Fresh frozen vege- 42 0 7 0 33 0

tables

Totals ......... 180 234 77 6 167 96

* Present in 0.1 g test portion.

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H. ZABOROWSKI, D. A. HUBER, AND M. M. RAYMAN

may be observed after 24 hr of incubation, it is advisableto keep the cultures at least 72 hr before discardingthem as negative. Confirmation in ethyl violet azidebroth, as described above, is required.

Quantitative tests. If quantitative determinations aredesired, a most probable number (MPN) method maybe employed using a series of consecutive decimal dilu-tions to growth extinction. For this purpose inoculate asuitable number of replicate tubes of azide glucosebroth with 1 ml amounts, and, from the significantnumber of confirmed tubes in each of the appropriatethree highest dilutions, calculate the numerical valueusing MPN tables (Buchanan and Fulmer, 1928).

(e) Salmonella. Inoculate 50 ml of a 1:5 suspension ofproduct (equivalent to 10 g of food) into 200 ml ofselenite F broth containing cystine. After 18 to 20 hrincubation, streak the cultures onto brilliant green andbismuth sulfite agar plates and examine as describedby Byrne et al. (1955).

SUMMARYSuitable bacteriological methods for the examination

of precooked frozen foods are discussed. Tests includestandard plate count, coliform plate count, detection ofcoagulase-positive staphylococci and of enterococci.

For convenience, the use of mechanical blenders toprepare samples for analysis is recommended as astandard procedure. For frozen meals, the entire frozenblock of meat or vegetable is blended to insure a repre-sentative sample.

Isolation of coagulase-positive staphylococci is facili-tated by an enrichment in trypticase soy broth contain-ing 10 per cent sodium chloride, followed by streakingon tellurite glycine agar.A comparison of several liquid media for the detec-

tion and enumeration of enterococci is described. Of themedia tested, enterococcus presumptive broth andazide dextrose broth, when followed by a confirmatorytest, are considered suitable for use in the examinationof frozen foods.

REFERENCES

American Public Health Association 1946 Report of theCommittee on Standard Methods for the MicrobiologicalExamination of Foods: Tentative methods for the micro-biological examination of frozen foods. Am. J. PublicHealth, 36, 332-335.

American Public Health Association 1953 Standard methodsfor the examination of dairy products, Ed. 10, New York,New York.

BUCHANAN, R. E. AND FULMER, E. I. 1928 Physiology andbiochemistry of bacteria. Vol. I, pp. 10-12. The Williams& Wilkins Co., Baltimore, Maryland.

BURTON, M. 0. 1949 Comparison of coliform and entero-coccus organisms as indices of pollution in frozen foods.Food Research, 14, 434-436.

BYRNE, A. F., RAYMAN, M. M., AND SCHNEIDER, M. 1). 1955MIethods for the detection and estimation of numbers ofSalmonella in dried eggs and other food products. Appl.Microbiol., 3, 368-372.

[VOL. 6

CHAPMAN, G. H. 1945 The significance of sodium chloridein studies of staphylococci. J. Bacteriol., 50, 201-203.

EVANS, J. B., BUETTNER, L. G., AND NIVEN, C. F., JR. 1950Evaluation of the coagulase test in the study of staphylo-cocci associated with food poisoning. J. Bacteriol., 60,481-484.

FITZGERALD, G. A. 1947 How to control the quality of frozencooked foods. Food Inds., 19, 623.

HAJNA, A. A. 1951 A buffered azide glucose-glycerol brothfor presumptive and confirmative tests for fecal strepto-cocci. Public Health Lab., 9, 80.

HAJNA, A. A. AND PERRY, C. A. 1943 Comparative study ofpresumptive and confirmative media for bacteria of thecoliform group and for fecal streptococci. Am. J. PublicHealth, 33, 550-556.

HARTMAN, G. 1937 Ein Beitrag zur Reinzuchtung vonMastiticstreptokoken aus verunreinigen Material. Milch-wirtsch. Forsch., 18, 116.

HILL, J. H. AND WHITE, E. C. 1929 Sodium chloride mediafor the separation of gram positive cocci from gram nega-tive bacilli. J. Bacteriol., 18, 43-57.

HUBER, D. A., ZABOROWSKI, H., AND RAYMAN, M. M. 1958Studies on the microbiological quality of precooked frozenmeals. Food Technol., 12, (4) In Press.

JONES, A. H. AND FERGUSON, W. E. 1951 A study of methodsof preparing food products for microbiological analyses.Food Research, 16, 126-132.

LARKIN, E. P., LITSKY, W., AND FULLER, J. E. 1955 Fecalstreptococci in frozen foods. II. Effect of freezing storageon Escherichia coli and some streptococci inoculated ontogreen beans. Appl. Microbiol., 3, 102-104.

LITSKY, W., MALLMANN, W. L., AND FIFIELD, C. W. 1955Comparison of most probable numbers of Escherichia coliand enterococci in river water. Am. J. Public Health,45, 1049-1053.

LOGAN, P. P., HARP, C. H., AND DoVE, W. F. 1951 Keepingquality of precooked frozen chicken a la king, a bacteriolog-ical evaluation of hot and cold packs. Food Technol.,5, 193-198

MAITLAND, H. B. AND MARTYN, G. 1948 A selective mediumfor isolating staphylococcus based on the differentialinhibiting effect of increased concentrations of sodiumchloride. J. Pathol. Bacteriol., 60, 553-561.

MALLMANN, W. L. AND SELIGMANN, E. B. 1950 A compara-tive study of media for the detection of streptococci inwater and sewage. Am. J. Public Health, 40, 286-289.

MOORE, B. 1955 Streptococci and food poisoning. J. Appl.Bacteriol., 18, 606-618.

RAYMAN, M. M., HUBER, D. A., AND ZABOROWSKI, H. 1955Current microbiological standards of quality for pre-cooked frozen foods and their basis. In Precooked F-ozenFoods-a Symposium. Advisory Board on QuartermasterResearch and Development, Committee on Foods, Na-tional Academy of Sciences, National Research Couincil,Washington, D. C.

ROTHE, W. C. 1948 Personal communication to the DifcoLaboratories. Difco Manual. Ed. 9, p. 148. Detroit,Michigan.

SHERMAN, J. M., MAUER, J. C., AND STARK, P. 1937 Streptococcus fecalis. J. Bacteriol., 33, 275-282.

WINTER, C. E. AND SANDHOLZER, L. A. 1946 Studies of thefecal streptococci. I. The isolation of enterococci fromnatural sources. Fishery Leaflet 201. U. S. Fish andWildlife Service.

ZEBOVITZ, E., EVANS, J. B., AND NIVEN, C. F., JR. 1955

Tellurite-glycine agar: A selective plating medium forthe quantitative detection of coagulase-positive staphy-lococci. J. Bacteriol., 70, 686-690.

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