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Journal of Food Protection, Vol. 56, No. 8, Pages 666-671 (August 1993) Copyright©, International Association of Milk, Food and Environmental Sanitarians

Evaluation of Stabilized Bismuth Sulfite Agar for Detection of Salmonella in Foods

SUSAN B. ALLEN, RUTH FIRSTENBERG-EDEN*, DEBORAH A. SHINGLER, CHRISTOPHER B. BARTLEY, and NADINE M. SULLIVAN

DIFCO R&D Center, 1180 Ellsworth Road, Ann Arbor, Michigan 48108 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021

(Received for publication December 22, 1992)

ABSTRACT a greenish tinge, which results in a less inhibitory medium for most species of Salmonella (4). McCoy (7) noted that proper The combination of a new manufacturing process, packaging, aging of BSA is essential for production of typical Salmo­ and media engineering has all contributed to the development of nella colonies in 18 h, whereas freshly poured BSA usually extended shelf-life (stabilized)-plated media (DuraPak™). One of require 48 h of incubation for blackening of colonies. D'Aoust the first products stabilized by this process was bismuth sulfite agar plates (BSA). Stabilized BSA were stored 3-4 months at 35°C and (5) observed that incubation of freshly prepared BSA for 48 then were inoculated in parallel with lab-prepared BSA following h resulted in good recovery of Salmonella and effective overnight aging at room temperature. Several strains of Salmonella inhibition of nonsalmonellae. Plates stored even 1 d at refrig­ were tested in pure cultures and in foods, including raw ground erated temperature did not inhibit Citrobacter freundii, which turkey, chicken, and nonfat dry milk. Also, two naturally contami­ occasionally mimics Salmonella on BSA. Andrews et al. (1) nated raw ground turkey and chicken samples were tested. Both observed some loss of selectivity after 2 d of storage. Earlier lab-prepared and stabilized BSA detected all positive samples after papers (4,7) did not explore the inhibition of non-Salmonella 24 h incubation at 35°C. Recovery, colony size, and amount of sheen produced by Salmonella were generally equivalent on both when evaluating aged plates. Thus, the use of BSA appears types of BSA, but stabilized BSA often yielded enhanced blacken­ to require a compromise. If consistent 24 h detection of ing of colonies and of the surrounding medium. Results were Salmonella is desired, the plates must be aged and some dependent upon strain of Salmonella used, type of food, and type inhibition of nonsalmonellae must be sacrificed. of enrichment broth streaked onto the plates. Stabilized BSA were Maximum shelf life of prepared BSA is generally esti­ generally more inhibitory to nonsalmonellae than lab-prepared mated to be < 5 d (2,4,5); storage beyond this point results in BSA, as demonstrated with pure cultures and food samples. Over­ significant medium degradation, indicated by a green color all, the performance of stabilized BSA was equivalent to that of lab- prepared BSA. and unsatisfactory growth and selectivity (4,5). The short shelf life of BSA necessitates frequent preparation in the laboratory. Consistency among batches of lab-prepared plates Bismuth sulfite agar plates (BSA) are valuable for the is often difficult to achieve (4,7) and requires monitoring the isolation of foodborne Salmonella because of their selectivity preparation and performance of each batch (1,3,6). The use of and ability to yield characteristic colonies. Most interfering commercially prepared medium, which is generally better organisms, including gram-positive microorganisms and col- controlled (1,6) with performance assured to the expiration iforms, are inhibited on BSA, while most salmonellae form date, eliminates the need for extensive testing; however, with characteristic green to black colonies surrounded by a brown- their extremely short shelf life, BSA have not been made black to black zone in the medium; by reflected light, this available commercially as a prepared plated medium. Re­ zone also exhibits a distinct metallic sheen (2). Because cently, a new plated media product (DuraPak; Difco Labora­ standard culture methods for the isolation of Salmonella tories, Detroit, MI) was developed through a delicate balance require several days to identify presumptive positive samples of storage atmosphere, manufacturing process, and protective (1,3,6), it is desirable that a medium, such as BSA, detect package barrier to yield plates with 6-12 month shelf life at Salmonella in 24 h rather than 48 h. room temperature (8). The use of DuraPak eliminates both Earlier studies have concluded that freshly prepared BSA the requirement of refrigerated storage and the need for (no aging prior to inoculation) can be inhibitory to salmonel­ frequent media preparation and extensive quality control lae other than Salmonella typhi. As a result, both recovery testing of laboratory-prepared media. and typical colony morphologies require 48 h of incubation This study was conducted to compare the perfor­ (4,5,7). Many sources (1,3,4,7) recommend that plates be mance of stabilized (DuraPak) BSA with that of lab- prepared in advance and allowed to age, usually 1-2 d at prepared BSA made from a commercial dehydrated me­ refrigerated or room temperature. The aging provides a slight dium using pure cultures and naturally and artificially oxidation of brilliant green and the sulfite, giving the medium contaminated foods.

JOURNAL OF FOOD PROTECTION, VOL. 56, AUGUST 1993 EVALUATION OF STABILIZED BISMUTH SULFITE AGAR 667 MATERIALS AND METHODS at room temperature overnight prior to their inoculation and incubated for 24 h thereafter. Foods Samples of nonfat dry milk (NFDM), raw whole chicken, raw Pure culture enrichment procedure cut-up chicken, and raw ground turkey were obtained from local Pure cultures of salmonellae were diluted to 10 CFU/ml in supermarkets and tested on the day of purchase. Foods initially peptone water and inoculated individually at 1:50 into tetrathionate determined by Association of Official Analytical Chemists/Bacte­ (TT) and selenite cystine (SC) broths to obtain 0.2 CFU/ml in riological Analytical Manual (AOAC/BAM) (1) methodology to be selective enrichment. The inoculated broths were mixed in a Salmonella free were kept refrigerated for 3-5 d for subsequent Stomacher 400 (Tekmar, Cincinnati, OH) for 2 min and then inoculated food studies. allowed to stand 1 h at room temperature, securely sealed. Broths were then mixed, and the pH was determined and adjusted to pH Reagents and media 6.8 ± 0.2, if needed, with 1 N NaOH or 1 N HC1. After the addition All reagents and media were as specified in AOAC/BAM (7) of 2.25 ml of 0.1% brilliant green dye solution to TT, the samples and were obtained from Difco Laboratories (Detroit, MI) unless were mixed thoroughly and incubated 24 + 2 h at 35°C. Broths otherwise noted. Fresh BSA plates were prepared 1 d prior to were then stomached for approximately 30 s and 3-mm loopfuls inoculation and were stored in the dark at room temperature were spread onto lab-prepared BSA, stabilized BSA, PCA, and overnight as recommended in AOAC/BAM (7). DuraPak stabilized xylose lysine desoxycholate agar (XLD). Plates were observed after Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021 BSA had been prepared from the same powder lot of bismuth 24 and 48 h incubation at 35°C. sulfite agar as the lab-prepared medium and packages of prepared For nonsalmonellae pure culture studies, the isolates were plates were stored for 3-4 months at 35°C. This storage period was diluted to 106 CFU/ml in 0.1% peptone water diluent and added at found to be equivalent to storage of 6-8 months at 25°C (unpub­ 1:10 into TT and SC to obtain 105 CFU/ml in selective enrichment. lished data). Prior to use, physical parameters of the stabilized BSA The inoculated broths were then treated as above. Growth on the which were evaluated included pH, color, opacity, and moisture plated media was examined after 24 and 48 h at 35°C. loss. Stabilized BSA plates were removed from their package and, like lab-prepared BSA, were stored overnight in the dark at room Food studies temperature. All Salmonella testing in foods (both artificially and naturally contaminated) was done substantially by AOAC/BAM (1) proce­ Test cultures and preparation of inocula dure for the particular food being tested. Raw ground turkey (25 g) Nine Salmonella strains were used to assess the performance was added to 225 ml of both TT and SC prior to stomaching. The of BSA with regards to growth and colony morphology. Two enrichment cultures were streaked onto stabilized BSA, lab-pre­ cultures (K-4 and M-13) were previously isolated from raw ground pared BSA, and XLD. Any suspect Salmonella colonies on these turkey and identified as Salmonella spp. by Gram stain, PASCO agar media were restreaked onto XLD and PCA. Isolates with Gram-Negative Panel (Difco Laboratories, Detroit, MI) and API typical Salmonella morphologies on these media were then Gram 20E (Analytab Products, Plainview, NJ). Serotyping by somatic stained. The identity of gram-negative rods with typical colony (O) antisera was done to help in presumptive identification: K-4 morphologies was further characterized by either PASCO Gram- and M-13 were positive by poly A and poly B antisera, respec­ Negative Panel or API 20E. The growth of non-Salmonella on lab- R tively. Additional Salmonella cultures were obtained from ATCC prepared and stabilized BSA after 24 and 48 h at 35°C was (American Type Culture Collection, Rockville, MD) with the compared. exception of Salmonella infantis CDC 906, which was obtained For studies involving raw chicken, the chicken was placed into from the Difco Laboratories stock culture collection. Eight species a sterile stomacher bag containing 100 ml sterile 0.1% peptone of non-Salmonella microorganisms were also isolated from ground water. The chicken was "massaged" with the diluent, and then the turkey. These isolates were identified by Gram stain, PASCO liquid was decanted. Ten milliliters of the liquid rinse was inocu­ Gram-Negative, and API 20E. Two Escherichia coli and two C. lated into 90 ml each of TT and SC, and the procedure for highly freundii strains which mimic salmonellae on BSA were obtained contaminated or raw meat products was again followed. from Dr. J.-Y. D'Aoust (Health Protection Branch, Health & For isolation of Salmonella from NFDM, a preenrichment of Welfare Canada, Sir Frederick G. Banting Research Centre, Tunney's 25 g NFDM in 225 ml 0.002% brilliant green water was incubated Pasture, Ottawa, Ontario, Canada KAOL2). 24 + 2h at 35°C. After mixing, 1-ml portions of preenrichment All cultures were maintained on refrigerated plate count agar were transferred to both 10 ml SC and 10 ml TT. Isolation of (PCA) slants. From the refrigerated slants, cultures were grown 18 Salmonella was then continued as above. h at 35°C on PCA plates. Multiple colonies were swabbed into and Inoculated samples of raw ground turkey, raw chicken, and then serially diluted in 0.1% peptone water to obtain the desired NFDM were also analyzed substantially by the AOAC/BAM pro­ inoculum level. For both pure culture and inoculated food studies, cedure. The Salmonella test strain was prepared in peptone diluent Salmonella strains were enumerated by inoculating 25 ul of the 103 4 and added to the preenrichment or enrichment broth along with the and 10 peptone dilutions in duplicate onto PCA and incubating for food being tested to a final concentration of 0.2 CFU/ml. The 24 h at 35°C. densities of Salmonella and nonsalmonellae on XLD, and on the stabilized and lab-prepared BSA plates, were estimated Evaluation of BSA performance semiquantitatively; suspect isolates were restreaked to XLD and When evaluating for Salmonella growth, stabilized and PCA. Typical morphologies on these three media were used as lab-prepared BSA were compared for time to recovery, colony culture confirmation of Salmonella. number and size, and amount of blackening and sheen pro­ duced. Both media were considered to be equal if the recoveries RESULTS AND DISCUSSION were within 25% of each other. Colony size and blackening were scored visually. For non-Salmonella, colonies on stabi­ lized and lab-prepared BSA were compared for degree of Overnight storage of BSA growth inhibition, colonial size, and colonial morphology (mim­ The appearance of stabilized and lab-prepared BSA was icry to Salmonella). Recoveries within 25% were considered to comparable. Initially, stabilized BSA were inoculated imme­ be equivalent. Results relate to BSA that were stored in the dark diately after opening the packages, but most Salmonella

JOURNAL OF FOOD PROTECTION, VOL. 56, AUGUST 1993 668 ALLEN, FIRSTENBERG-EDEN, SHINGLER, BARTLEY AND SULLIVAN strains required 48 h incubation for definitive blackening of incubation. In contrast to the findings of other authors (4,5), colonies and development of a metallic sheen. These results the recovery of typical Salmonella colonies did not increase compare to those seen with fresh lab-prepared and inoculated significantly with an additional day of incubation. Aging the BSA and agree with the observations of other authors (4,5,7). plates overnight at room temperature likely reduced the level Thus, packages of stabilized BSA were opened and stored at of inhibition to Salmonella. room temperature overnight in the dark prior to use, in Number and size of Salmonella colonies recovered were accordance with the AOAC/BAM (1) recommendation for generally equivalent on both types of BSA. Amount of sheen lab-prepared plates. During this storage, the BSA developed and blackening produced was enhanced on stabilized BSA. a faint greenish tinge, indicating slight oxidation of the However, this difference was not always observed on all brilliant green and sulfite (4). The lack of aging of lab- plates. Only one strain, S. infantis CDC 906, produced prepared and stabilized BSA delayed the growth of Salmo­ noticeably better results on lab-prepared BSA. nella; the delay was more pronounced on the stabilized BSA. In the second phase of the pure culture study, non- The need for an aging step to help lessen the inhibitory nature Salmonella microorganisms capable of growth in the enrich­ of the medium and allow for the production of typical ment broth and on BSA were isolated from ground turkey Salmonella colonies in 24 h has been previously noted (4,7). and identified. Pure cultures of these isolates and additional Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021 With 48 h incubation, the reactions on immediately opened E. coli and C. freundii strains which mimic salmonellae on and streaked stabilized plates were better than those on lab- BSA were also plated on both agar media following selective prepared plates. Colonies were generally larger and blacker enrichment in Salmonella broths. Performance of BSA was and often produced better sheen on the stabilized plates. evaluated according to the number of colonies recovered, size of colonies, and their ability to mimic Salmonella (Table 2). Pure culture studies Stabilized BSA were judged to perform better than or com­ Results from these studies (Table 1) show that perfor­ parable to lab-prepared BSA for all 12 isolates evaluated. In mance of stabilized and lab-prepared BSA was comparable. general, colony sizes of the nonsalmonellae were signifi­ Salmonella colonies were detected on both media after 24 h cantly smaller, and thus, less darkening (and sheen for some)

TABLE 1. Performance of stabilized and lab-prepared BSA with pure cultures of Salmonella.

Performance ratings

TT SC

Salmonella serotype CFW Recovery Colony Black­ Sheen Recovery Colony Black­ Sheen ml size ening size ening

S. saintpaul 0.01 _b = Sc S = S F" ATCC 9712

S. heidelberg 0.02 = = S S = = = ATCC 8326

S. typhimurium 0.30 = = S S = S = ATCC 14028

S. infantis 0.60 = F F F F = = F CDC 906

S. enteritidis 0.80 ======ATCC 13076

S. oranienburg 0.80 = = = S = S S ATCC 8238

Salmonella spp. 0.80 = = = = = F = K-4

Salmonella spp. 0.80 ======M-13

S. newport <1 ======ATCC 6962

CFU/ml inoculated into TT and SC. = - equal performance of stabilized and lab-prepared BSA. S - better performance by stabilized BSA. F - better performance by lab-prepared BSA.

JOURNAL OF FOOD PROTECTION, VOL. 56, AUGUST 1993 EVALUATION OF STABILIZED BISMUTH SULFITE AGAR 669

TABLE 2. Performance of stabilized and lab-prepared BSA, with pure cultures of non-Salmonella.

Performance ratings

TT SC

Culture CFUa/ Inhibition Decreased Colonial Inhibition Decreased Colonial ml of growth colony size distinctness of growth colony size distinctness

C. freundii D16 105 _b _ _ = — — 5 C.freundii D17 10 ======E. coli D12 105 oc 0 0 = = = H. alvei 105 = Sd s = S S K. oxytoca 105 0 0 0 = = = K. pneumoniae 105 = = 5 s s s s P. mirabilis 10 ======

5 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021 P. vulgaris 10 ======P. alcalifaciens 105 = = = = 5 s s P. rettgeri 10 s = = = s = P. stuartii 105 = = = 0 0 0

* CFU/ml inoculated into TT and SC. b = - equal performance of stabilized and lab-prepared BSA. c 0 - no growth in both types of BSA. d S - better performance by stabilized BSA.

TABLE 3. Performance of stabilized and lab-prepared BSA, in naturally contaminated foods.

Performance ratings

Enrichment Ground Ground Ground Whole Cut-up medium turkey K2 turkey B2 turkey M2 chicken chicken

Salmonella d Recovery =" n n = n Colony size = n n = n Blackening P n n S" n TT Sheen F n n F n

Non-Salmonella

Amount of = = t/ i = = inhibition Colonial = = F = F distinctness

Salmonella Recovery n n n n n Colony size n n n n n Blackening n n n n n SC Sheen n n n n n

Non-Salmonella Amount of = = = = = inhibition Colonial = = = S = distinctness

Salmonella Isolation yesc no no yes no

= - equal performance of stabilized and lab-prepared BSA. S - better performance by stabilized BSA. F - better performance by lab-prepared BSA. n - no salmonellae isolated. yes = isolated from TT on both stabilized and lab-prepared BSA: not isolated from SC.

was observed on stabilized BSA. This difference between an organism which had initially been isolated as a potential both types of BSA was particularly evident for Hafnia alvei, Salmonella on lab-prepared BSA. Colonies of this culture

JOURNAL OF FOOD PROTECTION, VOL. 56, AUGUST 1993 670 ALLEN, FIRSTENBERG-EDEN, SHINGLER, BARTLEY AND SULLIVAN were much smaller on the stabilized BSA. Overall, recovery taminated with Salmonella, which in each case was only for nonsalmonellae on both media was equivalent and un­ recovered from the TT enrichment. For these two samples, changed after 48 h incubation. As noted by D'Aoust (5), BSA recovery and size of Salmonella colonies were equivalent for stored for 1 d at room temperature is not inhibitory toward C. lab-prepared and stabilized plates. Colonies on lab-prepared freundii. In these experiments, recoveries of the two BSA gave better sheen reactions; blackening of colonies and Citrobacter strains on both lab-prepared and stabilized plates the surrounding medium was better on stabilized BSA for the were 75-100% of those obtained on PCA. whole chicken sample and better on lab-prepared BSA for the The results obtained with both types of BSA demonstrate ground turkey. However, even though the lab-prepared plates that a delicate balance exists in the bismuth sulfite agar were rated better for sheen and darkening for ground turkey, inhibitory system. If Citrobacter and other organisms that the Salmonella colonies on the stabilized BSA were easily mimic Salmonella need to be minimized, a higher degree of recognizable as Salmonella; the reactions observed for the inhibition is desired. However, the use of such media will two media were never drastically different. result in a delay in the development of typical Salmonella The amount of growth of nonsalmonellae and its resem­ colonies. To obtain characteristic Salmonella colonies in 24 blance to Salmonella colonies were also evaluated. In all h, packages of stabilized BSA must be opened 1 d in cases, whether Salmonella was present or not, both media Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021 advance, sacrificing some inhibition of non-Salmonella. were generally equivalent. No growth was observed from the NFDM sample enrichment, by either Salmonella or interfer­ Food studies ing organisms. Therefore, NFDM results are not included in Table 3 summarizes the results for Salmonella (recovery, Table 3. colony size, and degree of blackening and sheen) and for non-Salmonella (amount of inhibition and colonial distinct­ Inoculated food studies ness from Salmonella). Since time to recovery of Salmonella Four strains of Salmonella were tested: 2 ATCC strains was equivalent on stabilized and lab-prepared BSA, these and 2 which had previously been isolated from raw ground results were not included in the table. Two of six samples turkey. All four were tested in NFDM and raw ground turkey; (ground turkey K-2 and whole chicken) were naturally con­ all but S. enteritidis were also tested in chicken. Inoculum

TABLE 4. Performance of stabilized vs. lab-prepared BSA with Salmonella-inoculated foods.

Performance ratings

S. typhimurium S. enteritidis Salmonella sp. Salmonella sp. Enrichment ATCC 14028 ATCC 13076 K-4 M-13 Medium NFDM Turkey Chicken NFDM Turkey NFDM Turkey Chicken NFDM Turkey Chicken

Salmonella Recovery a ======Sb = = Colony size = = S = = = = Blackening S S s s S S TT Sheen S = = = s s s

Non-Salmonella Amount of n" = = n = n = s n = = inhibition Colonial n = n = n = s n = = distinctness

Salmonella Recovery ======Colony size = = = p = = = = Blackening = s S s = = s SC Sheen = F ======F

Non-Salmonella Amount of n = n n = n = = n = = inhibition Colonial n = n n = n = = n = = distinctness

= - equal performance of stabilized and lab-prepared BSA. S - better performance by stabilized BSA. F - better performance by lab-prepared BSA. n - no nonsalmonellae isolated.

JOURNAL OF FOOD PROTECTION, VOL. 56, AUGUST 1993 EVALUATION OF STABILIZED BISMUTH SULFITE AGAR 671 levels in the foods ranged from 2-8 CFU/g. As in the storage. One medium, tryptic soy agar with 5% sheep blood, naturally contaminated foods, Salmonella was recovered within has been stabilized for 8 months at 2-8°C (8). 24 h after incubation by both the stabilized and lab-prepared BSA. Based upon the performance parameters used, overall ACKNOWLEDGMENTS results were better on the stabilized BSA (Table 4). Stabilized We would like to thank Dr. J.-Y. D'Aoust and Ms. K. Flanagan for BSA plates inoculated from TT performed better than lab- providing cultures. prepared BSA plates similarly inoculated in 12 of 44 com­ parisons; equivalent performance was obtained in the remain­ REFERENCES ing 32 comparisons. In addition, stabilized BSA produced enhanced blackening of Salmonella colonies in 10 of 22 1. Andrews, Wallace H., P. L. Poelma, and C. R. Wilson. 1984. instances using TT or SC enrichment cultures; in no case did Isolation and identification of Salmonella species, pp. 7.01-7.18. In FDA Bacteriological Analytical Manual, 6th ed. Association of Offi­ the lab-prepared BSA perform better for this parameter. cial Analytical Chemists, Arlington, VA. Results comparing the two types of BSA for the 2. Anonymous. 1984. Difco manual, 10th ed. Difco Laboratories, Inc. detection of Salmonella in ground turkey were equivalent. Detroit, MI. In NFDM and chicken samples, stabilized BSA perfor­ 3. Anonymous. 1974. USDA microbiology laboratory guidebook. FSIS, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/56/8/666/1662331/0362-028x-56_8_666.pdf by guest on 28 September 2021 Microbiology Division, Beltsville, MD. mance was better than that of lab-prepared BSA for 18 of 4. Cook, G. T. 1952. Comparison of two modifications of bismuth- 84 Salmonella and nonsalmonellae comparisons under sulphite agar for the isolation and growth of Salmonella typhi and both enrichment conditions; only three of 84 comparisons Salm. typhimurium. J. Pathol. Bacterid. 64:559-566. were better with lab-prepared BSA (Table 4). Ten of the 5. D'Aoust, J.-Y. 1977. Effect of storage conditions on the performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124. 18 superior ratings on stabilized BSA were for enhanced 6. Flowers, R. S., J.-Y. D'Aoust, W. H. Andrews, and J. S. Bailey. blackening of Salmonella colonies and the surrounding 1992. Salmonella, pp. 371-422. In Vanderzant, C, and D. F. medium. Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods, 2nd ed. American Public Health Association, This study shows that the stabilized BSA (DuraPak) can Washington, DC. be utilized to detect Salmonella in foods, instead of lab- 7. McCoy, J. H. 1962. The isolation of salmonellae. J. Appl. Bacteriol. prepared plates. The utilization of DuraPak BSA will assure 25:213-224. consistent quality of plates which requires less quality control 8. Sullivan, N. M., C. B. Bartley, W. R. Avalone, D. A. Carlson, S. B. than laboratory-prepared plates. In addition to BSA, 30 other Leasor, and R. Firstenberg-Eden. 1991. Development of a manufac­ turing process for stabilizing plated media. Abstr. No. 1-105, pre­ plated media have been successfully stabilized in the DuraPak sented at 91st General Meeting, American Society for Microbiology, format to a shelf-life of at least 8 months room temperature Dallas, TX, May 5-9.

Kim, Slavic, Griff is, and Walker, cont. from p. 665

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