Vol. 43, No. 2 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1982, p. 261-268 0099-2240/82/020261-08$02.00/0 Rapid Detection of Salmonella spp. in Food by Use of the ISO-GRID Hydrophobic Grid Membrane Filter PHYLLIS ENTIS,l* MICHAEL H. BRODSKY,1 ANTHONY N. SHARPE,2 AND GEORGE A. JARVIS3 QA Laboratories Limited, Toronto, Canada M9C IC2'; Bureau of Microbial Hazards, Health Protection Branch, Health and Welfare Canada, Ottawa, Canada, KIA OL22; and Food Statistics and Operational Planning, Health Protection Branch, Health and Welfare Canada, Ottawa, Canada KIA IB83 Received 30 June 1981/Accepted 29 September 1981 A rapid hydrophobic grid-membrane filter (HGMF) method was developed and compared with the Health Protection Branch cultural method for the detection of Salmonella spp. in 798 spiked samples and 265 naturally contaminated samples of food. With the HGMF method, Salmonella spp. were isolated from 618 of the spiked samples and 190 of the naturally contaminated samples. The conventional method recovered Salmonella spp. from 622 spiked samples and 204 unspiked samples. The isolation rates from Salmonella-positive samples for the two methods were not significantly different (94.6% overall for the HGMF method and 96.7% for the conventional approach), but the HGMF results were available in only 2 to 3 days after sample receipt compared with 3 to 4 days by the conventional method. Government and industry standards for Sal- monella spp. in foods and animal feeds specify that this organism must be absent from as many as 60 25-g analytical units drawn from a produc- tion lot (15). To attain this level of sensitivity, it is usually recommended that an overnight non- selective enrichment followed by an overnight selective enrichment procedure be used (3). As a result, a conventional Salmonella spp. analysis requires a minimum of 3 days after sample receipt to obtain presumptive results (acceptable method MFA-20 of the Health Protection Branch of Health and Welfare Canada, Ottawa). Several researchers have developed screening procedures in an attempt to reduce the time required to complete a Salmonella spp. analysis. These methods include fluorescent antibody staining (18), enrichment serology (1, 19), en- zyme-linked immunology (11), and radiometry (20). All four procedues are carried out either directly from the selective enrichment broth or from a postenrichment broth which has been incubated for several hours. Little information is available to date on the reliability of the radiometry or the enzyme- linked immunology techniques; however, fluo- rescent antibody staining and enrichment serolo- gy are known to produce false-positive results (12). For example, false-positive rates of 4 to 17% have been reported in the literature for fluorescent antibody staining (7, 8, 14, 21, 22). It is essential, therefore, to confirm a presumptive screening result by isolating the Salmonella spp. in the conventional way. This requirement ne- gates some of the benefits of the screening procedures, especially when foods containing high levels of competing gram-negative bacteria are being evaluated. In 1957, Kenner et al. (9) developed a mem- brane filtration technique for the isolation of Salmonella spp. from water and feces. They employed a short (4 to 6 h) selective enrichment procedure, filtered the enrichment through a membrane filter, and incubated the membrane on a selective medium. Colonies which devel- oped on the membrane were then subcultured for conventional biochemical and serological evaluation. In 1962, Kirkham and Hartman (10) described a direct membrane filter method for the enumeration of Salmonella spp. in egg albu- men. They suggested that this method could be used in place of a most-probable-number meth- od. Recently, Brodsky et al. (M. H. Brodsky, P. Entis, M. P. Entis, A. N. Sharpe, and G. A. Jarvis, J. Food Prot., in press) reported on the use of the ISO-GRID hydrophobic grid-mem- brane filter (HGMF; QA Laboratories Ltd.) for several quantitative food microbiological analy- ses. The ISO-GRID membranes provided a counting range in excess of 3 logl0 cycles on a single filter and were very effective in restricting the lateral spread of virtually all microbial colo- nies. The hydrophobic property of the grid lines allowed the detection and enumeration of specif- ic microorganisms in the presence of a higher level of background flora than could be tolerated by conventional plating or membrane filtration procedures. This report describes a rapid membrane filtra- 261 on August 31, 2020 by guest http://aem.asm.org/ Downloaded from
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Vol. 43, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1982, p. 261-2680099-2240/82/020261-08$02.00/0
Rapid Detection of Salmonella spp. in Food by Use of theISO-GRID Hydrophobic Grid Membrane Filter
PHYLLIS ENTIS,l* MICHAEL H. BRODSKY,1 ANTHONY N. SHARPE,2 AND GEORGE A. JARVIS3QA Laboratories Limited, Toronto, Canada M9C IC2'; Bureau of Microbial Hazards, Health ProtectionBranch, Health and Welfare Canada, Ottawa, Canada, KIA OL22; and Food Statistics and Operational
Planning, Health Protection Branch, Health and Welfare Canada, Ottawa, Canada KIA IB83
Received 30 June 1981/Accepted 29 September 1981
A rapid hydrophobic grid-membrane filter (HGMF) method was developed andcompared with the Health Protection Branch cultural method for the detection ofSalmonella spp. in 798 spiked samples and 265 naturally contaminated samples offood. With the HGMF method, Salmonella spp. were isolated from 618 of thespiked samples and 190 of the naturally contaminated samples. The conventionalmethod recovered Salmonella spp. from 622 spiked samples and 204 unspikedsamples. The isolation rates from Salmonella-positive samples for the twomethods were not significantly different (94.6% overall for the HGMF method and96.7% for the conventional approach), but the HGMF results were available inonly 2 to 3 days after sample receipt compared with 3 to 4 days by theconventional method.
Government and industry standards for Sal-monella spp. in foods and animal feeds specifythat this organism must be absent from as manyas 60 25-g analytical units drawn from a produc-tion lot (15). To attain this level of sensitivity, itis usually recommended that an overnight non-selective enrichment followed by an overnightselective enrichment procedure be used (3). As aresult, a conventional Salmonella spp. analysisrequires a minimum of 3 days after samplereceipt to obtain presumptive results (acceptablemethod MFA-20 of the Health ProtectionBranch of Health and Welfare Canada, Ottawa).
Several researchers have developed screeningprocedures in an attempt to reduce the timerequired to complete a Salmonella spp. analysis.These methods include fluorescent antibodystaining (18), enrichment serology (1, 19), en-zyme-linked immunology (11), and radiometry(20). All four procedues are carried out eitherdirectly from the selective enrichment broth orfrom a postenrichment broth which has beenincubated for several hours.
Little information is available to date on thereliability of the radiometry or the enzyme-linked immunology techniques; however, fluo-rescent antibody staining and enrichment serolo-gy are known to produce false-positive results(12). For example, false-positive rates of 4 to17% have been reported in the literature forfluorescent antibody staining (7, 8, 14, 21, 22). Itis essential, therefore, to confirm a presumptivescreening result by isolating the Salmonella spp.in the conventional way. This requirement ne-gates some of the benefits of the screening
procedures, especially when foods containinghigh levels of competing gram-negative bacteriaare being evaluated.
In 1957, Kenner et al. (9) developed a mem-brane filtration technique for the isolation ofSalmonella spp. from water and feces. Theyemployed a short (4 to 6 h) selective enrichmentprocedure, filtered the enrichment through amembrane filter, and incubated the membraneon a selective medium. Colonies which devel-oped on the membrane were then subculturedfor conventional biochemical and serologicalevaluation. In 1962, Kirkham and Hartman (10)described a direct membrane filter method forthe enumeration of Salmonella spp. in egg albu-men. They suggested that this method could beused in place of a most-probable-number meth-od.
Recently, Brodsky et al. (M. H. Brodsky, P.Entis, M. P. Entis, A. N. Sharpe, and G. A.Jarvis, J. Food Prot., in press) reported on theuse of the ISO-GRID hydrophobic grid-mem-brane filter (HGMF; QA Laboratories Ltd.) forseveral quantitative food microbiological analy-ses. The ISO-GRID membranes provided acounting range in excess of 3 logl0 cycles on asingle filter and were very effective in restrictingthe lateral spread of virtually all microbial colo-nies. The hydrophobic property of the grid linesallowed the detection and enumeration of specif-ic microorganisms in the presence of a higherlevel of background flora than could be toleratedby conventional plating or membrane filtrationprocedures.
FIG. 1. Growth transfer device (ISO-GRID repli-cator).
tion method for the detection of Salmonella spp.which takes advantage of the beneficial proper-ties of the ISO-GRID HGMF to shorten the
selective enrichment procedure and to simplifythe isolation and biochemical screening of thesuspect Salmonella spp.
MATERIALS AND METHODS
ISO-GRID HGMFs and filtration units. ISO-GRIDHGMFs and filtration units were produced by QALaboratories as described elsewhere (P. Entis, M. H.Brodsky, and A. N. Sharpe, J. Food Prot., in press).Growth transfer device (ISO-GRID replicator). The
ISO-GRID replicator (Fig. 1) was designed and builtby QA Laboratories (patent pending). The unit con-sists of a velvet ribbon for transferring growth and an
alignment mechanism for ensuring that colonies aretransferred onto the corresponding grid coordinates ofeach replicate ISO-GRID membrane. The alignmentdevice is composed of a Bausch & Lomb stereoscopepod, model Stereo 1, and a mechanical stage. Align-ment is effected by adjusting the stage to bring themarker on the ISO-GRID membrane into alignmentwith a cross hair in the stereoscope ocular.Media and reagents. The media developed for the
ISO-GRID HGMF procedure were formulated asshown in Table 1, sterilized by autoclaving at 121°C,and dispensed aseptically into petri dishes. The follow-ing culture media were prepared and sterilized accord-ing to the manufacturer's recommendations: bismuthsulfite agar (GIBCO Laboratories), brain heart infu-sion agar (GIBCO), brilliant green sulfa agar (DifcoLaboratories and GIBCO), lysine iron agar (Difco andGIBCO), nutrient agar (Difco), nutrient broth (Difco),selenite-cystine broth (Difco), tetrathionate broth base(Difco) and triple sugar iron agar (Difco). Brilliantgreen and iodine were added to tetrathionate broth
TABLE 1. Composition of selective media for ISO-GRID HGMF analysis
immediately before use as specified in Health Protec-tion Branch acceptable method MFA-20. Brilliantgreen water was also prepared as specified in MFA-20.
Preparation of inoculum for spiked samples. Thirty-eight Salmonella spp. serotypes (Table 2) were ob-tained from the Health Protection Branch for use inthis study and maintained on tryptic soy agar (Difco)slants at 5°C.
Cell suspensions of individual serotypes were pre-pared as described previously (Entis et al., in press).The actual inoculum level was determined by filtering10 ml of each suspension through an HGMF andincubating the filter on selective lysine agar at 35°C for24 ± 2 h. The number of occupied grid squares wascounted, and the count per milliliter was calculated byusing the formula described elsewhere (Brodsky et al.,in press). The range ofinoculum levels was found to be0.02 to 100 cells per 25 g.
Parallel analyses. Two versions of the HGMF meth-od (Figs. 2 and 3), were used in the parallel study andcompared against Health Protection Branch methodMFA-20. Whereas the HGMF methods differed fromeach other with respect to media formulations andsome incubation temperatures, both methods incorpo-rated the following stages: overnight nonselective en-richment, 6-h selective enrichment, filtration of selec-tive enrichment, overnight incubation of filter onprimary isolation medium, replication (if required)onto three secondary biochemical media, overnightincubation of secondary media, and subculture ofpresumptive salmonellae for serological confirmation.
Parallel analyses were carried out on approximatelytwo to six samples per lot as shown in Fig. 2 (method1) on 130 raw poultry samples, 187 raw ground meatsamples, 117 raw liver samples, 200 samples of nonfatdry milk, 170 dried foods samples (including a variety
TABLE 2. Distribution by food type of Salmonella spp. serotypes used to inoculate samples
lactose sucrose fermentation agar; SOR, sorbitol agar;ICA, iron cystine agar; BHIA, brain heart infusionagar; NA, nutrient agar.
of spices, chocolate products, cake mixes, and miscel-laneous low-moisture products), and 144 liquid eggsamples (both fresh shell eggs and frozen, pasteurizedegg). An additional 115 parallel analyses were carriedon raw poultry by using the modified procedure (meth-od 2) outlined in Fig. 3. All samples, with the excep-tion of the raw poultry, were preweighed into individ-ual sterile bags in 25-g amounts. Each sample was theninoculated with 0.1 ml of a freshly prepared Salmonel-la sp. suspension (Table 2) and stored at -18°C for twoto nine days to stress the organisms before proceedingwith the parallel analysis. The entire 25-g sample was
then analyzed.Interpretation of ISO-GRID growth pattern. The
ISO-GRID sample processor used in this study wasdescribed elsewhere (Brodsky et al., in press). As eachISO-GRID membrane was evaluated, the locations ofall colonies on the grid manifesting a biochemicalreaction consistent with Salmonella spp. were auto-matically stored. Once all four ISO-GRID HGMFsbelonging to a given sample had been so evaluated, thesample processor identified the grid coordinates ofpresumptive Salmonella spp. colonies.Three presumptive ISO-GRID biochemical profiles
for Salmonella spp. were recognized (Table 3). Up tothree colonies per sample were selected for serologicalscreening if "typical" colonies were identified by thecomputer. Up to five colonies were screened serologi-
C others
t35 C
FIG. 3. Parallel analysis procedure for evaluationof ISO-GRID Salmonella method 2. For the stepindicating "appropriate" volume, when testing raw,comminuted products, filter 10 ,ul (or 1 ml of a 10-2dilution); for all other products, filter 100 ,ul. Abbrevi-ations: TBG, tetrathionate broth; SLA2, modifiedselective lysine agar; LFA, lactose fermentation agar;SOR2, modified sorbitol agar; ICA, iron cystine agar;BHIA, brain heart infusion agar; NA, nutrient agar.
cally if only "lactose-positive" or "H2S-negative"colonies (or both) were found. Serological screeningwas carried out with Difco somatic antiserum, polyva-lent A-I+Vi.
Statistical evaluation of parallel analysis data. Thedata obtained for each product type were analyzed byusing a one-sided binomial test (13) to determine thestatistical significance of the number of disagreementsbetween the ISO-GRID and conventional methods.Where the number of disagreements was significant, atwo-sided McNemar test (17) was applied to determinewhether the distribution of the disagreements wassignificantly biased in favor of one or the other meth-od.
RESULTSThe results of parallel analyses carried out on
1,063 food samples are summarized in Table 4.Salmonella spp. were recovered by at least onemethod from 641 of the 798 spiked samples and213 of the 265 naturally contaminated samples.The overall isolation rate for the ISO-GRIDHGMF method was 94.6% as compared with96.7% for the conventional method. In foodscontaining relatively few organisms capable ofgrowth in the selective media, namely, nonfatdry milk, egg products, and dried foods, theisolation rates were 98.5 and 96.5% for theHGMF and conventional methods, respectively.The data obtained for each type of product wereevaluated by using a one-sided binomial test.The number of disagreements between the ISO-
a As determined on selective lysine agar or modified selective lysine agar; lysine-positive colonies are blue;ysine-negative colonies are yellow or yellow-green.b As determined on m-lactose sucrose agar, lactose fermentation agar, or modified lactose fermentation agar;
lactose-positive colonies are blue; lactose-negative colonies are colorless to beige.As determined on sorbitol agar or modified sorbitol agar; sorbitol-positive colonies are blue; sorbitol-
negative colonies are colorless to beige.d As determined on iron cystine agar; H2S-positive colonies are black; H2S-negative colonies are yellow or
beige.
GRID and conventional methods were statisti-cally insignificant at the 95% confidence levelfor dried foods, egg products, nonfat dry milk,and raw liver. The data from the two remainingtypes offood, raw ground meat and raw poultry,in which the number of disagreements was sig-nificant, were subjected to a two-sided McNe-mar test. The results of this test showed that nosignificant difference existed at the 95% confi-dence level in the number of false-negative re-sults produced by either method.To determine whether the ISO-GRID proce-
dure performed equally well at both high andlow levels of Salmonella spp. contamination, theresults of the parallel analyses carried out on 798spiked samples were sorted by inoculum level.The ISO-GRID method was as effective at in-oculum levels below 1 per 25 g as at higher levelsof inoculum (Table 5).A supplementary study was performed to
evaluate the reliability of the biochemical pro-files obtained with method 2. Of the 427 rawpoultry samples included in the study, 285 weredetermined by the ISO-GRID sample processorto contain typical Salmonella spp. A total of 763
typical HGMF isolates were obtained from the285 samples (up to 3 isolates per sample) andwere tested serologically with Salmonella so-matic polyvalent antiserum. A total of 742(97.3%) of the 763 isolates agglutinated with theSalmonella antiserum. A total of 282 (99%) ofthe 285 samples yielded at least one typicalHGMF isolate which agglutinated with the anti-serum.
Since organisms other than Salmonella spp.can agglutinate with the somatic polyvalent anti-serum, 271 of the 742 isolates which had aggluti-nated were further characterized by their reac-tion patterns in triple sugar iron agar and lysineiron agar. All 271 isolates produced reactionstypical of Salmonella spp.An additional study was carried out concur-
rently to determine the frequency at which thepresence of Salmonella spp. would not havebeen detected on the ISO-GRID membrane ifthe sample processor had been limited to atypical profile. Eighty-four samples of raw poul-try in which no biochemically typical isolateshad been detected by the sample processor wereexamined by inoculating up to five lactose-
TABLE 4. Recovery of Salmonella spp. by ISO-GRID HGMF and conventional methodsNo. of samples
ProducttypePositiveby Positive by Negative byProduct type Positive by Positive by Positive by HGMF and HGMF andTetd either HGMF MFA-20 negative by positive bymsenlethod HGMF MFA-20
a Each sample was individually inoculated with asingle Salmonella sp. serotype and analyzed by meth-od 1.
b Numbers within parentheses indicate the percent-age of positive samples.
positive or H2S-negative (or both) colonies fromeach of these samples into triple sugar iron agarand lysine iron agar and by testing the isolateswith Salmonella somatic polyvalent antiserum.This study was initially carried out by HGMFmethod 2 (Fig. 3). Biochemically typical salmo-nellae were recovered from 23 samples (13.9%of the total number of positive samples detectedduring the study). Since nearly all of theseisolates had been identified by the sample pro-cessor as lactose-positive, method 2 was subse-quently modified slightly by replacing lactosefermentation agar with modified lactose fermen-tation agar to improve the reliability of thelactose fermentation reaction, and the study wasrepeated with 58 samples. By using modifiedlactose fermentation agar, biochemically typicalSalmonella spp. isolates were recovered fromonly two samples (1.4% of the total number ofpositive samples detected during this phase ofthe study) from which the ISO-GRID sampleprocessor had not detected typical salmonellae.
DISCUSSIONConventional methods for isolating Salmonel-
la spp. rely, in part, on the use of highly selec-tive enrichment conditions and plating media todetect Salmonella spp. in the presence of highlevels of competing organisms. In order to iso-late a Salmonella spp. biotype which may beinhibited by these selective conditions, mostconventional methods specify the use of preen-richment, two selective enrichment broths, anda minimum of two plating media (5, 16; HealthProtection Branch method MFA-20). To simpli-fy the HGMF method, we elected initially to usea single primary isolation medium of relativelylow selectivity (selective lysine agar). Althoughthis approach (method 1) produced excellentresults when samples containing relatively lowbackground flora were analyzed, we experi-enced heavily saturated primary isolation mem-branes when analyzing raw meats (liver, poul-try, and ground meat). Although the level of
background flora in these foods did not preventthe detection of Salmonella spp., it did presentsome difficulty in interpreting the biochemicalprofile and, consequently, pecessitated the sub-culturing of a larger number of isolates thanwould be desirable. We therefore reevaluatedand revised our initial procedure to reduce thelevel of background flora without reducing thesensitivity of the method (method 2).The decision to use a 1:100 ratio for the
inoculation of the selective broth and to performonly a single selective enrichment on tetrathion-ate broth at 35°C for method 2 was based on theresults of a series of experiments which demon-strated that these enrichment conditions werethe most appropriate to use with the ISO-GRIDmembrane (unpublished data). Inoculation of 10ml of selective enrichment with 0.1 ml of over-night nonselective broth has been reported in theliterature to be as effective as the more tradition-al procedure (3) and, when Rappaport's selec-tive enrichment broth was used, the optimalinoculum/broth ratio for Salmonella spp. recov-ery was as low as 1:1,000 (6). Modificationswere also made in the formulations of most ofthe plating media to improve the growth ofSalmonella spp. on the primary medium and todiscourage the growth of nonsalmonellae onboth the primary and the secondary media. Thismodified procedure (method 2) was then evalu-ated in a parallel study of 115 samples of natural-ly contaminated raw poultry. The introductionof method 2 did not adversely affect Salmonellaspp. recovery by the ISO-GRID approach (Ta-ble 4). This modified procedure produced pri-mary HGMFs with a much lower level of back-ground flora than had been produced withmethod 1.
Lysine-negative Salmonella spp. variants areknown to occur and have been found principallyin the serovars Salmonella cholerae-suis andSalmonella paratyphi A (4). These two serovarsaccounted for only 0.3% of human Salmonellaspp. isolations in Canada during the first 6months of 1980 (Canada Diseases Weekly report7(8):39, 1981). Due to the very low reportedincidence of lysine-negative salmonellae, thisatypical reaction was not included in the ISO-GRID sample processor biochemical profile. Al-though the sample processor is capable of de-tecting lactose-positive and H2S-negativeSalmonella spp. biotypes, our studies haveshown that such designations must be treated aspresumptive, and a thorough biochemical andserological evaluation of these isolates should becarried out.The Salmonella spp. method which evolved
as a result of these studies is summarized inTable 6. Since the performance of this methodwith raw poultry was at least as good as the first
TABLE 6. Suggested ISO-GRID HGMF procedurefor detection of Salmonella spp.
Step Procedure
1 Perform an overnight (18- to 24-h) nonselectiveenrichment at 35°C using the appropriate brotha
2 Transfer 0.1 ml of the nonselective broth into 10ml of TBG; incubate at 35°C for 6 to 8 h
3 Filter the appropriate quantityb of tetrathionatebroth through an ISO-GRID HGMF; place onmodified selective lysine agar and incubate 24 ±2 h at 430C
4 Read the HGMF with the ISO-GRID sampleprocessor; if no blue colpnies are detected, theanalysis is complete; if blue colonies aredetected, replicate onto sterile ISO-GRIDHGMFs on iron-cystine agar, modified lactosefermentation agar, and modified sorbitol agar;incubate for 18 to 24 h at 35, 43, and 35°C,respectively
5 Read the HGMFs; perform the computerizedevaluation of the four HGMFs
6 If required, subculture for serologicalconfirmation
a As described in MFA-20.b When testing raw, comminuted products, filter 10
,ul (or 1 ml of a 10-2 dilution); for all other products,filter 100 p1l.
HGMF method tested, we believe that this latestversion of the method will be very effective inrecovering Salmonella spp. from foods withlower levels of competing background flora.This conclusion should, of course, be validated,ideally by an independent, multilaboratory col-laborative study.The rapid Salmonella spp. method described
in this paper has an important advantage overother presently available screening methods. Incontrast to procedures such as fluorescent anti-body techniques or enrichment serology, thedetection of typical Salmonella spp. colonies is areliable indicator of the presence of Salmonellaspp. in a sample. Due to the very low false-positive rate associated with the detection ofbiochemically typical colonies by the sampleprocessor in the latest version of the method, nofurther confirmation is necessary for most rou-tine applications, unless the investigator re-quires serological characterization for an epide-miological study. Even then, subculture of theisolate is a simple matter, since its exact locationon the ISO-GRID membrane is known. Con-versely, with the exception offoods which favorthe development of atypical biotypes (2), thenondetection of typical Salmonella spp. coloniesis a reliable indicator of the absence of Salmo-nella spp. in a sample. Thus, the ISO-GRIDprocedure is a dependable, rapid, stand-alonemethod for the routine screening of foods andtheir ingredients for Salmonella spp.
ACKNOWLEDGMENT
This project was carried out under Contract ISZ79-00130 ofSupply and Services Canada.We are grateful to P. Boleszczuk, H. Shannon, and J.
Muscat for their suggestions and their excellent technicalassistance. We thank Michael P. Entis for designing theprototype equipment used in this study. The comments andsuggestions of J. Y. D'Aoust are greatly appreciated.
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