APPLED MICINOLOGY, Apr. 1972, p. 679-682Copyright 0 1972 American Society for Microbiology

Vol. 23, No. 4Printed in U.SA.

Salmonella Testing of Pooled Pre-EnrichmentBroth Cultures for Screening Multiple Food

SamplesW. R PRICE, R. A. OLSEN, ArD J. E. HUNTER

Winton Hill Technical Center, The Procter & Gamble Company, Cincinnati, Ohio

Received for publication 10 December 1971

A method has been described for testing multiple food samples for Salmo-nella without loss in sensitivity. The method pools multiple pre-enrichmentbroth cultures into single enrichment broths. The subsequent stages of theSalmonella analysis are not altered. The method was found applicable to sev-eral dry food materials including nonfat dry milk, dried egg albumin, cocoa,

cottonseed flour, wheat flour, and shredded coconut. As many as 25 pre-en-richment broth cultures were pooled without apparent loss in the sensitivity ofSalmonella detection as compared to individual sample analysis. The proce-dure offers a simple, yet effective, way to increase sample capacity in the Sal-monella testing of foods, particularly where a large proportion of samples ordi-narily is negative. It also permits small portions of pre-enrichment broth cul-tures to be retained for subsequent individual analysis if positive tests are found.Salmonella testing of pooled pre-enrichment broths provides increased consu-mer protection for a given amount of analytical effort as compared to individualsample analysis.

Positive test results are relatively rareamong the many samples of food ingredientsand products which are analyzed for Salmo-nella. Therefore, a test procedure that gives asingle negative answer for many negative sam-ples offers an important saving in the analyt-ical effort required for quality control. Thesestudies support the conclusion that Salmonellapre-enrichment broths can be pooled for anal-ysis without loss in sensitivity to Salmonellain the individual samples.

MATERIALS AND METHODSSalmonella analyses were carried out as described

in the Bacteriological Analytical Manual (BAM) (2)except for the pooling of pre-enrichment broths de-scribed in the following paragraph.The procedure for Salmonella testing of pooled

pre-enrichment broths is illustrated in Fig. 1. Sam-ples are individually pre-enriched followed bytransfer from multiple pre-enrichments to single sel-eni'e and tetrathionate broths at the enrichmentstage. During pooling, a portion of each pre-enrich-ment is transferred to a sterile culture tube and re-tained at 4 C for later reference to the individualsamples. If the Salmonella test of the pooled pre-enrichment broths is positive, individual tests on theretained samples can be made to determine which

sample or samples contributed Salmonella to thepool.

Pre-enrichment broths consisted of 0.5% lactosebroth for all foods except nonfat dry milk, for whichsterile distilled water containing 0.002% BrilliantGreen was employed.

Salmonella-positive test samples were preparedby blending the dry test food with one of three dryinocula. Inoculum 1 was a dry enzyme drain cleanerwhich had been found to contain multiple Salmo-nella serotypes including E,,z1o; Gz19 (S. cubana);and C, g complex. Inoculum 2 was a freeze-dried,skim milk suspension of serotypes S. anatum, S.binza, S. tennessee, S. Worthington, S. cabana, S.braenderup, and a B1,g complex. Inoculum 3 con-sisted of four laboratory isolates of serotypes G,z,,(S. cabana); E1,b; E1,l complex; and C1,l complexgrown in a sterile aqueous suspension of cottonseedflour and freeze dried.

RESULTS AND DISCUSSIONDried egg albumen, cocoa, and nonfat dry

milk were inoculated with inoculum 1 at levelsranging from 6 salmonellae per 100 g to 3,000salmonellae per g. Pre-enrichment broths ofthe Salmonella-inoculated samples were ana-lyzed both individually and after pooling with5, 9, and 14 pre-enrichments of uninoculated

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Ls 0 00 0 f

FIG. 1. Salmonella testing ot.pooled pre-enrichment broths.

TABLE 1. Effect of pooling pre-enrichment broth cultures on Salmonella recovery from food samples

Inoculation Nonfat dry milk Cocoa Dried egg albuminlevela(per g) PI' P+5N P+9N P+14N P P+5N P+9N P±+14N P P+5N P+9N P+14N

Series I600 + + + + + + + + +60 + + + - -- + + +6 + + + - -- + + +0.6 + + + - -- + + +0.06

Series II3,000 + + + + + +

20 + + + + + +0.2 + + + + + +

a The most-probable-number estimate (1) of the original Salmonella inoculum (inoculum 1 containing sero-types Ezlo; G,z29; and C,g complex) was 93,000/g with 95% confidence limits ranging from 15,000 to380,000/g. The inoculation level represents a suitable dilution of inoculum 1, into an otherwise Salmonella-negative sample, to provide the levels as shown.

b Abbreviations: P, positive sample inoculated with naturally contaminated dry enzyme drain cleaner; N,negative (uninoculated) sample; +, Salmonella recovery positive; -, Salmonella recovery negative.

samples. The positive inoculated samples were

detected in pooled pre-enrichment broth cul-tures as effectively as when they were analyzedindividually (Table 1). The lvel of inoculation

affected the recovery of Salmonella equally forboth individual and pooled analyses. Detectionof Salmonella in cocoa was less sensitive thanin the other two test materials. A possibly sim-

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VOL. 23, 1972 SALMONELLA TESTING OF POOLED PRE-ENRICHMENT BROTHS

ilar antimicrobial effect of cocoa on strains of S.gallinarum and S. typhimurium was reportedby Busta and Speck (3).

Similarly, Salmonella was detected in wheatflour, coconut, and cottonseed flour sampleswithout loss in sensitivity, with both inoculum1 and inoculum 2, when single pre-enrichmentswere pooled with from 9 to 24 pre-enrichmentsof uninoculated samples.Pooled samples versus pooled pre-enrich-

ment broths. Silliker (4) reported a loss insensitivity when several samples were pooledin a large container of pre-enrichment broth ascompared to individual sample analysis. Thisis not surprising since, to be detected, the des-iccated and debilitated Salmonella cells in a

dried food sample must grow out in pre-en-richment broth in competition with other bac-teria in the sample. If many food samples arepooled at the pre-enrichment stage, the salmo-nellae face competition from the flora of allthe samples in the pool. If, on the other hand,the samples are individually pre-enriched, theactively growing salmonellae might be ex-pected to compete more favorably with orga-nisms introduced from other samples whenpooled at the enrichment stage. In addition,pooling pre-enrichment broths avoids the haz-ards and inconvenience of handling very largeflasks of culture.A study was made of Salmonella detection

in pooled samples and pooled pre-enrichmentcultures in the presence of added interferingorganisms, i.e., non-Salmonella bacteriahaving colonies that resemble Salmonella onthe selective agar media. The study was car-ried out with nonfat dry milk and egg albl-min, using inoculum 3. About 500 cells each offour interfering organisms, Pseudomonas sp.,Proteus sp., a lactose-positive Citrobacterfreundii, and a lactose-negative Bethesda-Bal-lerup were added at the pre-enrichment stage.

Pooled samples were prepared by placingnine 25-g Salmonella-negative samples andone 25-g inoculated sample together in a singlejar containing 2,250 ml of pre-enrichmentbroth. A pool of 10 samples was considered tobe a practical maximum for convenient han-dling. Pre-enrichment pooling was carried outas previously shown in Fig. 1 except that 1positive and 14 negatives were included in thepool.Table 2 compares pooling of samples and of

pre-enrichment broth cultures for nonfat drymilk and dried egg albumin at six levels ofSalmonella inoculation. The results show thata higher number of positives was recovered bypooling pre-enrichments rather than by pool-

TABLE 2. Effect of pooling stage on Salmonelladetectiona

Nonfat dry milk Dried egg albuminPooled Pooled

Pooled pre-en- Pooled pre-en-samplesc rich- samplesc rich-

Inoculation" (No. mentsd (No. mentsdlevel (per g) positive (No. positive (No.

3 repli- positive 3 repli- positivecates) 3 repli- cates) 3 repli-

cates) cates)

840 3 3 3 3420 3 3 3 317 2 3 3 38 2 3 0 10.33 3 2 0 10.07 0 3 0 2

Total 13/18 17/18 9/18 13/18a Approximately 500 cells each of Pseudomoras

sp., Proteus sp., Citrobacter freundii, and Bethesda-Ballerup were added to the pre-enrichment broth ofeach procedure.°The most-probable-number estimate of Salmo-

nellk in inoculum 3 (containing serotypes G,Z29; El,b; E1,L complex; and C1,L complex) was 2,800/gwith 95% confidence limits ranging from 1,000 to15,000/g. The inoculation level represents a suitabledilution of inoculum 3 into an otherwise negativesample to provide the levels as shown.

c Pooled samples = one positive and nine negativesamples.

d Pooled pre-enrichments = pre-enrichmentbroths from 1 positive sample and 14 negative sam-ples.

ing samples. In addition, pre-enrichment pool-ing . lded positives at lower levels of inocula-'on. Similar results were found in tests with-almonella-inoculated cocoa, shredded coco-nut, and wheat flour.The pooling of broth cultures permits small

portions of each individual culture to be re-tained for subsequent analysis if a positive testis detected following enrichment, thus main-taining the individuality of each test sample.On the other hand, the direct pooling of foodsamples into pre-enrichment results in the lossof individual sample identity.Number of isolates examined. Two or

more colonies are picked from each positiveselective plate for biochemical and serologicaltesting by the BAM Salmonella method (2) toincrease the chances of detecting Salmonella.By following this procedure, 180 isolates

could be tested if 15 samples were tested indi-vidually, whereas only 12 isolates could betested at the same rate of picking from a poolof 15 samples.To test the importance of the reduced

number of isolates, 238 isolates were picked at

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the rate of one colony per positive selectiveplate from the pooled pre-enrichment culturetests of the three food materials: dry milk, egg

albumin, and cocoa. Forty-four per cent of theisolates were Salmonella. In a similar evalua-tion of the tests on flour and coconut, 60% of72 isolates picked at the rate of two coloniesper plate were Salmonella. The high propor-

tion of Salmonella indicated that pooled pre-

enrichment provided an adequate number ofisolates to detect the presence of Salmonella.Whether pooled pre-enrichments are less

sensitive than individual tests in the presence

of interfering organisms probably is notknown. For the present, we would recommendthat, if numerous non-Salmonella colonies thatresemble Salmonella on selective agar mediaare encountered in the pooled test, the re-

tained pre-enrichment cultures should betested individually. Alternatively, problemswith interfering organisms may warrant theuse of direct selective enrichments, two selec-tive enrichments, or possibly even a reductionin the incubation time of the pre-enrichmentcultures.

Effect of volume of pre-enrichment brothtransferred. The BAM method (2) for Salmo-nella detection in egg products calls for thetransfer of 1 ml of pre-enrichment broth cul-ture to 10 ml of enrichment broth. In the mul-tiple pooling of pre-enrichment broth culturesthat has been described, 1-ml quantities of cul-tures were transferred, generally, to a volumeof enrichment broth determined by thenumber of samples to be pooled to maintain a

fixed pre-enrichment-to-enrichment ratio.Thus, 150 ml of enrichment broth was used toreceive 15 1-ml poolings, 100 ml for 10 1-mlpoolings, etc. However, for routine laboratoryoperations, it is more desirable to dispenseenrichment broth at a constant volume. Sincethe number of samples to be pooled may vary

considerably from time to time, it is importantto know whether variations in enrichment ratiowould significantly affect the detection ofSalmonella.

Accordingly, a study was undertaken to testthe effect of 1/10, 1/50, and 1/100 enrichmentratios on the recovery of Salmonella in inocu-lated food systems. The study included evalua-tion for both pooled samples and pooled pre-

TABLE 3. Effect of enrichment ratio on Salmonelladetection in inoculated food materiolsa

No. positive/18tests

Method of detection Enrichment ratio

1/10 1/50 1/100

Pooled pre-enrichments ....... 11 13 15Pooled samples .........9....._ 9 9

a Chi-square tests and tests for linear trend be-tween the number of positive tests and the enrich-ment ratio indicated no significant differences at theP = 0.05 level.

enrichment broth cultures. Each pooled pre-enrichment test was comprised of 15 25-gsamples, whereas each pooled sample test con-sisted of 10 25-g samples. Six different testswere included for each ratio. Table 3 summa-rizes the results of this study.No significant differences in detection were

noted among the three ratios tested as meas-ured by the chi-square test and the test forlinear trend. The tentative conclusion drawn isthat the enrichment ratio over the range inves-tigated and for the food samples tested (nonfatdry milk, dry egg albumin, and cocoa) was notcritical to Salmonella detection.We have also examined the effect of 1-ml

and 2-ml transfer volumes of the pre-enrich-ment cultures on Salmonella detection andfound no significant differences in recoveries.This finding has prompted the standard use ofa 1-ml transfer for routine Salmonella anal-yses.

LITERATURE CITED1. American Public Health Association. 1965. Standard

methods for the examination of water and wastewater,12th ed. American Public Health Association, NewYork.

2. Anonymous. Bacteriological analytical manual. Dept, ofHealth, Education, and Welfare, Food and Drug Ad-ministration. 1969.

3. Busta, F. F., and M. L. Speck, 1968. Antimicrobial effectof cocoa on salmonellae. Appl. Microbiol. 16:424-425.

4. Silliker, J. H. 1969. The influence of sample size on ana-lytical sensitivity in the analysis of dried yolk andwhole egg for salmonellae, p. 203-205. In An evaluationof the Salmonella problem. National Academy of Sci-ences publication no. 1683.

au802 APPL. MICROBIOL.

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