APPLIED MICROBIOLOGY, Apr. 1969, p. 533-539Copyright @ 1969 American Society for Microbiology

Vol. 17, No. 4Printed in U.S.A.

Accelerated Procedure for Salmonella Detection inDried Foods and Feeds Involving Only Broth

Cultures and Serological Reactions1W. H. SPERBER AND R. H. DEIBEL

Department of Bacteriology and The Food Research Institute, The University of Wisconsin,Madison, Wisconsin 53706

Received for publication 16 December 1968

A procedure has been developed in which Salmonella can be detected in driedfoods and feeds within 50 hr. This includes preenrichment (18 hr), selective enrich-ment (24 hr), elective enrichment (6 to 8 hr), and serological testing (2 hr). The pro-

cedure is as sensitive as the more time-consuming, traditional (plating, biochemical,and serological) procedure which may require at least 4 to 5 days. The new pro-cedure is rapid and accurate in comparison to traditional procedures. Moreover, itis simple and inexpensive to perform. As described, the procedure does not necessi-tate the isolation of pure cultures, but this step is performed easily if desired.

The food and feed industries are currently sub-jecting their products to an intensive scrutiny forSalmonella contamination. The recall of contam-inated product from the market, actual loss ofproduct, marred product prestige, and economicconsequences of increased control programs haveand will continue to have serious ramifications inthe affected industries.One of the major problems confronting in-

dustry today is the long time needed to derminewhether a product is free of Salmonella contami-nation. An absolute minimum of 4 days is re-quired (2), and, if the presumptive test is positive,an additional 2 to 3 days are necessary. In general,4 to 6 days must be allotted for this determination.This forces the manufacturer to consider two al-ternatives. One is to ship the product withoutwaiting for laboratory clearance; the other, towarehouse the product until laboratory clearanceis obtained. Neither alternative is economicallyappealing. In the first alternative, microbiologicalsafety can be violated and the product may besubject to recall or seizure. The other alternativeinvolves costly additional handling and storage ofproduct. These considerations lay the foundationfor one of the food and feed industries' mostpressing needs: an accelerated procedure forSalmonella detection.The accelerated procedure described herein

necessitates both preenrichment and selectiveenrichment by the same methods as practiced in

I Published with the approval of the Director of the WisconsinAgricultural Experiment Station.

the traditional procedure. However, instead ofsubsequently plating in selective agar media toeffect isolation followed by biochemical screeningand ultimate serological confirmation, the pro-posed procedure involves a 6-hr incubation in anelective broth medium and direct serologicaltesting. For the most part, the definitive identifi-cation of salmonellae is serological, and, thesooner these specific immunochemical reactionscan be employed in a given procedure, the lesstime will be required for its completion.

MATERIALS AND METHODS

Samples. Only foods and feeds that were con-taminated naturally with salmonellae were used inthis study. Most of these samples were suppliedthrough the generosity of various food companies.Also, some samples were obtained from the Food andDrug Administration Laboratories. Some animal feedsamples used in preliminary experiments were sup-plied by J. H. Silliker, Chicago Heights, Ill. Toenhance longevity of the contaminating organisms,all samples were stored in plastic containers at 4 C.

Preenrichment culture procedures. Usually, 100 g ofsample was added to 1 liter of Lactose Broth (Difco)as recommended by North (6). In some instances, theamount of sample examined was decreased [as withdried egg samples or in the most-probable-numbers(MPN) procedure], but the 10% (w/v) ratio of sampleto Lactose Broth was maintained. Some samples,such as chocolate, that contained large amounts ofwater-insoluble material, were mixed with the lactosebroth in a mechanical blender. The preenrichmentcultures were usually incubated for 18 hr at 37 C.

Selective enrichment. Tubes containing 9 ml of

533

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

SPERBER AND DEIBEL

either Selenite-Cystine Broth (Difco) or TetrathionateBroth (Difco) were inoculated with 1-ml volumes ofthe preenrichment broth. These media were preparedwith only 90% of the recommended amount of waterso that, when the 1 ml of inoculum was added, theenrichment components were present at full strength.After shaking to insure distribution of the inoculum,the cultures were incubated at 37 C for 24 hr.

Traditional cultural procedure. Essentially, themethod of the U.S. Food and Drug Laboratorieswas employed (2). Dried plates of Salmonella-Shigella(SS) Agar (Difco), Brilliant Green Agar (Difco), andBismuth Sulfite Agar (Difco) were inoculated bystreaking one loopful of the selective enrichmentculture. Each enrichment was streaked on all threemedia. Thus, each sample ultimately required a totalof six plates. Sucrose at 1% strength was added to theSS Agar to differentiate sucrose-fermenting colonieson the plate. Also, 0.65% agar was added to SS Agarto obtain a firm medium with superior streakingcharacteristics. The inoculated plates were incubatedat 37 C for 24 hr, whereupon two to four suspectcolonies from each plate were picked to slants ofTriple Sugar Iron (TSI) Agar (Difco). The slants wereincubated overnight at 37 C. Presumptively Sal-monella-positive cultures were transferred into BrainHeart Infusion (BHI; Difco) and incubated at least8 hr at 37 C, or until densely turbid, for flagellar anti-gen tests using pooled (8) Spicer-Edwards antisera(Difco). The remaining growth on the TSI slant wasused for somatic (0) antigen analysis if the culturegave a positive test with pooled Spicer-Edwards anti-sera. If no reaction was obtained with Spicer-Edwardsantiserum, a polyvalent 0 test was performed. If theculture was also polyvalent 0-negative, it was notconsidered to be a Salmonella. To avoid overlookingmutant salmonellae, serological tests were performedon all TSI slants which were H2S-positive or whichexhibited an alkaline slant and acid butt. This proce-dure yielded two H2S-negative salmonellae whichotherwise would have been ignored.

RESULTSSpicer-Edwards test modification. Traditionally,

2.0 ml of a Formalin-salt mixture (0.6 ml Forma-lin, 0.85 g of NaCl, and 100 ml of water) is addedto 2.0 ml of a BH1 culture which has grown to therequisite turbidity. After gentle mixing, 0.5 ml ofthis mixture is placed in a Kahn tube (10 by 75mm) and 0.5 ml of diluted (1:500) Spicer-Edwards antiserum is added. Thus, in this test,the original culture is diluted fourfold but, sincepure cultures are used, this has never been amatter of importance.To circumvent the dilution of the original cul-

ture and thereby increase the sensitivity of thetest, the following modifications were employed.(i) The volume of culture was increased by dis-pensing 0.85 ml of the turbid broth into Kahntubes (10 by 75 mm). (ii) The concentration ofthe Formalin-salt solution was increased. Onedrop, or about 0.05 ml (3.0 ml of Formalin, 4.2 g

of NaCl, 100 ml of water), was added to the cul-ture in the Kahn tube. (iii) A more concentratedSpicer-Edwards antiserum preparation was em-ployed. The Spicer-Edwards antiserum wasdiluted 1:100 (in contrast to 1:500) and 0.1 mlwas added to the Formalin-culture mixture.The tubes were incubated at 50 C for at least 1

hr before being observed for agglutination. Purecultures of salmonellae grown in BHI or theTween 80-containing medium, APT Broth (Difco),were tested by the traditional and modified tests.With the traditional test, 2 X 108 cells/ml oforiginal culture were necessary for agglutination.In comparison, only 5 X 107 cells/ml of originalculture were necessary to give a positive resultwith the modified test. (This latter value is similarto the minimum number of cells necessary to givea positive reaction by fluorescent microscopy.)All subsequent flagellar agglutination tests wereperformed by using the modified test.

Spicer-Edwards tests with enrichment cultures.Attempts to shorten the traditional Salmonellaisolation procedure involved the coupling of themodified Spicer-Edwards test directly to the brothcultures used in Salmonella isolation, therebyeliminating the array of plating and biochemicaltest media necessary to identify presumptivesalmonellae. Neither the traditional nor themodified Spicer-Edwards test gave accurate re-sults when cultures of either enrichment mediumused in this study were examined. In some in-stances, this could be associated with the carry-over of precipitates to the agglutination tests(CaCO3 from Tetrathionate broth or seleniumfrom selenite-Cystine broth). Another and some-what common occurrence was the absence ofagglutination which perhaps reflects poor antigendevelopment in these relatively toxic media. Theseresults indicated the necessity of a subsequentgrowth period in a medium that favors antigen orflagellar development.

Testing various postenrichment media. BHI wasused in preliminary trials since this mediumaffords good antigen development for use in thetraditional Spicer-Edwards test. Although resultswith BHI were far superior to direct testing ofenrichment cultures, one false-negative result wasobtained when 73 samples were tested by theaccelerated procedure (results compared withtraditional cultural procedure). Moreover, diffi-culty was experienced in the agglutination tests, asspontaneous agglutination in the absence ofantisera was observed occasionally. Often theseresults could be associated with a "rough" orsedimentary type of growth in the broth culture.In an attempt to avoid this type of growth, someexperiments were undertaken with APT broth.Equivocal results were obtained; however, the

534 APPL. MICROBIOL.

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

ACCELERATED SALMONELLA DETECTION

high concentration of glucose (1.0%) in themedium could have afforded a radical drop in pHvalue. Presumably, by omitting the glucose andallowing citrate in the medium to serve as theanaerobic energy source, it was speculated that asuperior medium for Salmonella enrichmentmight result.The results with BHI and APT broth cultures

were not favorable. The use of APT broth minusglucose (APTMG) decreased the incidence ofnonspecific agglutination, but enrichment cul-tures from 3 of 73 samples still gave false-positiveor ambiguous results (controls spontaneouslyagglutinated).

In the course of this study, Old et al. (7) re-ported that fimbrial agglutination in Salmonellacultures could be eliminated by the addition ofmannose to the medium. In the hope that theseresults might extend to other Enuerobacteriaceae,and perhaps decrease further the incidence ofnonspecific agglutination in the modified aggluti-nation test, we added 0.2% D-mannose toAPTMG broth.

Preliminary results with key samples, knownto have caused difficulty previously, gave un-equivocal results. An extended study using themannose-contaiming medium (henceforth re-ferred to as "M broth" for simplicity) wasinitiated. Various dried food and feed sampleswere cultured in preenrichment and enrichmentmedia as described previously. Each enrichmentmedium was then further analyzed by the tradi-tional (plating-biochemical-serological) and theaccelerated enrichment serology (ES) proce-dures. In each of 105 individual tests (210 selec-tive enrichment cultures), an absolute correlationbetween the two test procedures was obtained(Table 1). Significantly, 37 of the test samplescontained Salmonella and some samples con-tained relatively low numbers (yeast powder anda food plant environmental sample) as evidencedby the frequency of positive results.A number of MPN determinations were made

on the milk chocolate, dried milk, and rosemaryspice samples (Table 2). The sensitivity of bothprocedures was equivalent, since identical MPNvalues were obtained for each food tested. Ifan enrichment culture was positive by oneprocedure, it was positive by the other and,conversely, if an enrichment culture was negativeby one procedure, it was also negative by theother.

Early in this phase of the study it was observedthat a one-drop inoculum from the selectiveenrichment culture to M broth and 6 to 8 hr ofincubation gave comparable results to a loopinoculum and overnight incubation. Most of thesamples (Table 1) were tested by both of these

TABLE 1. Comparison of traditional and ES pro-cedures for the detection of Salmonella in dried

foods and feedsSalmonella detection

results

No. of No. of Sam- Tradi- Tradi-Product lots ple Trd- raiotsples size ptioe; ngtive;

ES- ES-positivea negativea

gRosemary

spice........ 8 8 100 4 4Dried milk 9 9 100 5 4Egg albumen 1 11 10 3 8Food plantenviron-mentalsample. 1 20 10 1 19

Milkchocolate. 1 2 100 1 1

Yeast powder 1 10 10 2 8Gelatin.5 .......5 10 10 0 10Egg noodles 8 25 100 11 14Fish meal. 10 10 50 10 0Total 44 105 37 68

a Results obtained by each test procedure werethe same for each individual sample.

TABLE 2. Comparison of MPN results for Salmo-nella quantitation in dried foods using the

traditional and ES detection procedures

Product No. of lots MPN of salmonellaeexamined per 100 g of producta

Rosemary spice........ 8 0.36 to 4.3b(1 .7)c

Dried milk............ 1 9.3Milk chocolate ........ 16 0.36 to 2.3

(0.78)

a Three-tube MPN.b Identical results obtained with each procedure.c Arithmetic mean in parentheses.

procedures; the procedures gave identical results.Thus, if time is a critical factor and scheduleslend themselves to the shorter incubation period,results can be obtained somewhat earlier (about50 hr; otherwise, 3 days with the overnightincubation period in M-broth).

Effect of medium and other organisms. Through-out the course of this study, experiments wereperformed to determine the effect of relative num-bers of competing organisms on the performanceof the modified H agglutination test. Under actualtest conditions (i.e., after preenrichment andselective enrichment), it was estimated that anaverage (or perhaps minimal) inoculum from the

VOL. 17, 1969 535

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

SPERBER AND DEIBEL

selective enrichment broth would contain about103 Salmonella cells per ml. Thus, the minimalinoculum level in the M broth would be ap-

proximately 100 Salmonella cells per ml. Theseconditions were approximated by inoculating a

constant level of 106 non-Salmonella cells perml, varying the numbers of Salmonella cells (from106/ml to less than 100/ml) into M broth, andincubating for 18 hr. For comparative purposes,the three broth media described previously were

used. After incubation, the modified H-aggluti-nation test was employed for Salmonella detec-tion. Both APTMG and M broth affordedSalmonella detection at all levels of inoculation(Table 3). At the lowest levels of Salmonellainoculation (about 100 cells per ml), the maxi-mum ratio of non-Salmonella to Salmonellacells was approximately 10,000:1. Although bothAPTMG and M broth were effective equally,the use of APTMG was negated because ofoccasional spontaneous agglutination reactionswhen food samples were examined. Thus, rela-tively small numbers of Salmonella can be de-tected in the presence of overwhelming numbersof other Enterobacteriaceae.M-broth cultures and reactions with somatic

antisera. Some of the M-broth cultures ultimatelyderived from samples that were naturally con-

taminated with Salmonella were screened bythe slide agglutination test with somatic groupingantisera. One drop of the turbid M broth was

mixed with one drop of antiserum on a micro-scope slide, rotated, and observed in the usual

manner. Although extensive testing of thisprocedure was not attempted, its possible ap-

plicability is demonstrated in Table 4. In eachinstance, the group reactions obtained with M-broth preparation agreed with pure culturereactions obtained by the traditional platingprocedures. In each sample, only one serologicalgroup of contaminating Salmonella was en-

countered. If multiple 0 groups were present,then corresponding reactions with the respectivegroup antiserum would be expected.Recommended ES procedure for Salmonella

detection. (i) A 10% suspension of the food sam-ple is prepared in sterile Lactose Broth and in-cubated at 37 C for 18 to 24 hr.

(ii) One milliliter of the preenrichment cultureis transferred to 9 ml of Selenite-Cystine Brothand 1 ml to 9 ml of Tetrathionate Broth. Theenrichment media are incubated at 37 C for 24 hr.

(iii) M broth is prepared by adding 1.0 g ofTryptone (Difco), 0.5 g of Yeast Extract (Difco),0.5 g of NaCl, 0.5 g of K2HPO4, 0.5 g ofsodium citrate, 0.2 g of D-mannose, 2.0 ml ofsalts C, and 0.75 ml of 10% Tween 80 (Baker) to100 ml of distilled water. The pH is adjusted to7.0 with several drops of 4 to 6 N HC1 prior tosterilization. A 10-ml amount of the medium isdispensed into screw-cap tubes (16 by 125 mm)and autoclaved at 121 C for 15 min.

Salts C is prepared by dissolving 4.0 g ofMgSO4-7H20, 0.2 g of NaCl, 0.2 g of FeSO4V7H20, and 0.72 g of MnCl2.4H20 in 100 ml ofdistilled water. The stock solution is stored in a

TABLE 3. Effect of competing organisms and broth composition on the detection of S. thompson bya modified Spicer-Edwards test

Compteing organisms and broth culture median

S thompson/ml E. coli P. vulgaris Citrobacter sp.

BHI APT APTMG M BHI APT APTMG M BHI APT APTMG M

1,000,000 +b + + + + + ± + + + + +10,000 + - + + + - + + - - + +5,000 c 6 + + + - + + - - + +2,000 - - + + + - + + - - + +1,000 - - + + + - + + - - + +200 - - + + + - + + - - + +83 - - + + + - + +62 + - + +

Negative control,' - - - - - - - - - - - -Positive control, + + + + + + + + + + + +

aApproximately 1,000,000 cells/mi of the competing organism were inoculated into each tube.b Positive modified Spicer-Edwards test.¢ Negative modified Spicer-Edwards test.d Pure culture of competing organism.e Pure culture of S. thompson.

536 APPL. MICROBIOL.

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

ACCELERATED SALMONELLA DETECTION

TABLE 4. Somatic antigen grouping of salmonellae in M broth

Product

Rosemary spice..................Dried milk

(samples 1, 4, 5).............Dried milk

(samples 2, 7).................Egg albumen....................Milk chocolate

(samples 2, 4, 5, 6, 7).........Milk chocolate

(sample 8).....................Egg noodles

(samples 3, 4, 6)...............Egg noodles

(sample 2).....................

a No agglutination.I Agglutination.

refrigerator. A slight precipitatestorage and must be resuspendedadded to the medium.

Somatic antigen group

A B Ci

_+b

+

+

forms uponbefore being

The 10-ml volume of broth facilitates not onlythe employment of a drop inoculum from theenrichment broths, but also the subsequentwithdrawal of culture for serological tests suchthat no precipitates are included and activelymotile cells are obtained more readily. Thecitrate in M broth has a dual function in that itchelates the inorganic salts and also affords en-richment of Salmonella by serving as an energysource.

In this laboratory, preenrichment cultures areprepared before 3:00 PM each day. On the fol-lowing (second) morning, enrichment culturesare then inoculated at 8:00 to 9:00 AM. Thispermits at least an 18-hr incubation of the pre-enrichment culture. The M broth is inoculatedwith one drop of enrichment culture at 8:00 to9:00 AM on the next (third) morning, shaken todistribute the inoculum, tempered, incubated for6 hr at 37 C, and tested by the modified H-agglutination test. The M-broth cultures maybe incubated overnight at 37 C if the modifiedagglutination test cannot be performed on thesame day. In this event, the M broth should beinoculated with one loopful (0.01 ml) of theenrichment culture instead of one drop (0.05 ml).The primary purpose of inoculating the enrich-ment broths and M broth at 8:00 to 9:00 AM isto facilitate completion of the H-agglutinationtests at the end of the day.

(iv) The modified H-agglutination test is per-formed as follows. To one drop of Formalin-

C, D El E2 E4 F G H I

+

+

+

+

+

salt solution (3.0 ml of Formalin, 4.2 g of NaClin 100 ml distilled water) in a Kahn tube (10 by75 mm) is added 0.85 ml of M-broth culture.It is important not to agitate the M broth priorto removal of the sample, since any precipitatespresent (e.g., CaCO3 carried over from theTetrathionate Broth) might be resuspended andcause confusion in the reading of the agglutina-tion test. To this mixture is added 0.1 ml ofpooled antisera (see below). The Kahn tube isgently shaken and incubated at least 1 hr at50 C before observing for agglutination. Thesame criteria are employed as in reading othertube agglutination tests for H antigens in that anyvisible amount of floccular precipitate is regardedas a positive test for Salmonella.The pooled antiserum is prepared by adding

0.5 ml of each properly rehydrated Difco Hantiserum (Spicer-Edwards sera 1, 2, 3, 4, Lcomplex, en complex, 1 complex; and H antiseraZ6, poly D, and poly F) to 11.5 ml of 0.85%NaCl. The diluted serum is stored in a refrigeratorfor a maximum of 5 days before use. Extendedstorage of the preparation may be possible, butit was not attempted in this study.

It is imperative to temper all media to 37 Cbefore inoculation. This is particularly criticalwith the M broth, which is usually incubatedonly 6 hr.The M-broth culture can also be used to de-

termine the somatic antigens of salmonellaepresent. One drop of culture is mixed with onedrop of antiserum on a glass slide, gently rotated,and observed for agglutination. It will be neces-sary to agitate the M-broth culture if the cells

VOL. 17, 1969 537

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

SPERBER AND DEIBEL

have settled out by the time these determinationsare done. In this event, any CaCO3 or otherprecipitates present must be allowed to resettlebefore a portion of the culture is removed.

Loopfuls of the M-broth culture can also bestreaked onto appropriate selective agar mediumto isolate the salmonellae in pure culture, ifdesired.

DISCUSSION

To be totally acceptable, any improvedSalmonella detection procedure must not onlyfacilitate a decreased time factor but it must alsobe at least as sensitive and accurate as pro-cedures currently in use. The ES procedureproposed herein fulfills these criteria.An outstanding merit of the ES procedure is

that it can be performed easily at minimal cost.In both the traditional and ES procedures, thesample must be cultured in preenrichment andselective enrichment broths. It is well knownthat the many subsequent bacteriological manipu-lations in the traditional procedure are econom-ically undesirable, but a definitive cost estimateis difficult to provide. However, in the ES pro-cedure, an estimate can be made because theantisera constitute the major cost factor. Foreach H-agglutination test performed the cost ofthe antisera is approximately 5.5 cents. Thus,the economics associated with the ES procedureare extremely favorable not only in regard tocost of materials used in testing but also insavings associated with reduced laboratory laborand reduced product-warehousing costs.Some shortcomings of minimal magnitude are

inherent in the described ES procedure. Althoughmost of the agglutination tests reported in thisstudy were performed by using pooled Spicer-Edwards antisera, in the later phases the Hantigens detectable were expanded by inclusionof additional H antibodies. According to theKauffmann-White schema (4), 37 of 959 Sal-monella sp. cannot be detected by the pooledSpicer-Edwards antisera. However, only 3 ofthe 959 species will not be detected by pooledSpicer-Edwards antisera supplemented with thecommercially available H antisera z6, polyvalentD, and polyvalent F (Difco).The few samples examined indicated that the

supplemented pooled Spicer-Edwards antiseraneither aided nor detracted in the incidence ofpositive samples in comparison to unsupple-mented pooled Spicer-Edwards antisera. Al-though definitive data is not available from thisstudy to justify completely the inclusion of thethree additional antisera, it is deemed prudent toobtain the additional coverage.

The three species not detectable by the sug-gested pooled antisera are S. agona (group B),S. quinhon (group X), and S. pullorum-gallinarum(group D). In the 3-year period, 1965 to 1967,S. agona and S. quinhon were never isolated fromnonhuman sources and only one isolation wasmade from human sources (5). Moreover, therelative and real incidence of the avian pathogen,S. pullorum-gallinarum, appears to be steadilydecreasing (Table 5). Thus, the major factorthat cannot be evaluated with the statistical dataavailable is the incidence of nonmotile variants.However, even in instances where motility can-not be detected, positive reactions with H anti-sera have been observed (1). Although it isestimated that these isolates are relatively rare,definitive data regarding their occurrence islacking.

Previous studies have dealt with the utilityof using flagellar antisera for rapid screeningmethods of pure Salmonella cultures. Years ago,Hajna and Damon (3) pioneered H agglutina-tion as a rapid screening method for Salmonelladetection. More recently, Silliker et al. (8)reaffirmed the usefulness of this procedure. TheFood and Drug Administration Laboratoriesadvocate the employment of both a polyvalentH and Spicer-Edwards H antisera in their screen-ing procedure (9). However, all of these screen-ing methods embody isolation of colonies onselective agar media (24-hr growth period)followed by biochemical screening in TSI Agar(24 additional hr) and ultimately serologicaltests employing 6- to 8-hr broth cultures. Inessence, the ES procedure herein described obvi-ates the bacteriological manipulations and re-duces the time factor by 48 hr. With the accuracyand sensitivity of both procedures being equiva-lent, the economic benefits are in favor of theES procedure.

Preliminary studies indicate that the ES pro-cedure can be extended to other types of foodsaside from dried foods. Also, it is anticipatedthat the procedure will have utility in detecting

TABLE 5. Incidence of S. pullorum-gallinarum fromnonhuman sources3Total S. Total nonhuman .ulom-a-

Year pullorum- Salmonella S. oullorum-gall-Year lnaru isolations inarum isolationsisolations

1964 270 5461 5.01965 237 6864 3.'1966 80 7709 1.(1967 54 8794 0.6

a Annual Salmonella surveillance reports (5).

538 APPL. MICROBIOL.

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

ACCELERATED SALMONELLA DETECTION

Salmonella in fecal specimens. These studies willconstitute the subject of a subsequent report.

ACKNOWLEDGMENTS

The excellent technical assistance of R. M. Julseth and VickiWesen is gratefully acknowledged.

This investigation was supported by Hatch Act grant 1516 andby contributions from the food industry to the Food ResearchInstitute, University of Wisconsin, Madison.

LITERATURE CITED

1. Edwards, R. P., A. B. Moran, and D. W. Bruner. 1946. Fla-gella and flagellar antigens in "nonmotile" Salnonellacultures. Proc. Soc. Exptl. Biol. Med. 62:296-298.

2. Elliott, R. P. (ed.). 1966. Bacteriological analytical manual.Par. 14, Salnonella. U.S. Food and Drug Administration,Washington, D.C.

3. Hajna, A. A., and S. R. Damon. 1950. Polyvalent Salnonella"H" agglutination as a rapid screening test for Salmonellaorganisms. Public Health Rept. 65:116-118.

4. Kauffmann, F. 1966. Bacteriology of Enterobacteriaceae. TheWilliams & Wilkins Co., Baltimore.

5. National Communicable Disease Center. 1967. Salmonellasurveillance reports. Annual summaries 1965-1967. At-lanta.

6. North, W. R., Jr. 1961. Lactose pre-enrichment method forisolation of Salnonella from dried egg albumen. Its use in a

survey of commercially prepared albumen. Appl. Micro-biol. 9:188-195.

7. Old, D. C., I. Corneil, L. F. Gibson, A. D. Thomson, and J.P. Duguid. 1968. Fimbriation, pellicle formation and theamount of growth of salmonellas in broth. J. Gen. Micro-biol. 51:1-16.

8. Silliker, J. H., P. T. Fagan, J. Y. Chiu, and A. Williams. 1965.Polyvalent H agglutination as a rapid means of screeningnon-lactose-fermenting colonies for Salmonella organisms.Am. J. Clin. Pathol. 43:548-554.

9. Wazensli, J., K. J. Rhodes, and E. C. Robinson. 1968. Noteon the simultaneous use of polyvalent H and Spicer-Ed-wards H antisera as a preliminary step in the identificationof salmonellae. J. Assoc. Offic. Agr. Chemists 51:718-719.

VOL. 17, 1969 539

on June 8, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from