Immuno-detection and differentiation of Listeria monocytogenes and Listeria ivanovii in stone fruits.

AIMS: The aim of this study was to develop a rapid detection and differentiation method for pathogenic Listeria species in stone fruits. METHODS AND RESULTS: We utilized activated charcoal enrichment media (ACM) to induce overexpression and hypersecretion of pathogenic Listeria virulence proteins which can subsequently be detected via immunoblot analysis. Plum and nectarine slices spiked with either L. monocytogenes or L. ivanovii were incubated in pre-enrichment broth followed by enrichment in ACM. Secreted proteins were precipitated and subjected to SDS-PAGE and immunoblot analysis using a combination of L. monocytogenes-specific antibody (α-listeriolysin O) and antibody specific for both L. monocytogenes and L. ivanovii (α-Internalin C). As low as 1 CFU per gram of L. monocytogenes in plum and nectarine was detected, whereas a detection limit of 10 CFU per gram was achieved for L. ivanovii in each food tested following a 20-h enrichment period. Nonpathogenic Listeria species and non-Listeria bacterial pathogens tested were negative. CONCLUSIONS: These results demonstrate the highly sensitive and specific nature of the detection method for pathogenic Listeria in stone fruits using activated charcoal enrichment as well as the capability to discriminate between L. monocytogenes and L. ivanovii. SIGNIFICANCE AND IMPACT OF THE STUDY: This method is the first to identify and differentiate L. monocytogenes and L. ivanovii in select stone fruit enrichments within 24 h using immunological techniques. The rapidity and sensitivity of the method could aid in the reduction of exposure to the public in the event of an outbreak and expedite the administration of appropriate antibiotics to infected individuals.

Bio-Plex suspension array immuno-detection of Listeria monocytogenes from cantaloupe and packaged salad using virulence protein inducing activated charcoal enrichment media.

Listeria monocytogenes, the causative agent of listeriosis in humans, is a Gram-positive bacterium that is contracted via the ingestion of contaminated foods. Two of the largest outbreaks of listeriosis occurred following consumption of tainted cantaloupe and packaged salads. Molecular methods and immuno-based techniques for detection of L. monocytogenes in these food matrices can be difficult due to the presence of assay inhibiting elements. In this study, we utilized a novel enrichment media containing activated charcoal as the key ingredient that induces hyperactive expression and secretion of L. monocytogenes virulence proteins. The Bio-Plex suspension array system, based on Luminex xMAP technology, was subsequently employed to specifically detect accumulated L. monocytogenes secreted and membrane bound proteins via paramagnetic microsphere-antibody complexes. Cantaloupe and packaged salad samples were treated with a dilution series of L. monocytogenes and incubated in activated charcoal media following a short pre-enrichment step in Buffered Listeria Enrichment Broth. Secreted L. monocytogenes lysteriolysin O was captured using magnetic microsphere-antibody conjugates and measured using the Bio-Ple×200 analyzer. As few as 100 CFU/g of L. monocytogenes was detected from both spiked cantaloupe and packaged salad samples. In addition, antibody conjugated microspheres targeting a membrane protein present on both pathogenic and nonpathogenic Listeria species was used to identify as few as 100 CFU/g of both pathogenic and nonpathogenic species in cantaloupe and packaged salad. This method presumptively identifies L. monocytogenes from cantaloupe and packaged salad in less than 24 h and non-pathogenic Listeria species within 22 h.

Evaluation of 3M Molecular Detection System and ANSR Pathogen Detection System for rapid detection of Salmonella from egg products.

Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310-1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre-enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre-enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW-based culture method results. When LB was used as pre-enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre-enrichment broth.

Comparison of different preenrichment broths, egg:preenrichment broth ratios, and surface disinfection for the detection of Salmonella enterica ssp. enterica serovar Enteritidis in shell eggs.

Salmonella enterica ssp. enterica serovar Enteritidis is the leading reported cause of Salmonella infections. Most Salmonella Enteritidis infections are associated with whole shell eggs and egg products. This project attempted to lay the foundation for improving the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella Enteritidis in shell eggs. Two Salmonella Enteritidis isolates were used for comparisons among different preenrichment and enrichment media and for the evaluation of egg:preenrichment broth ratios for the detection of Salmonella Enteritidis in shell eggs. The effect of surface disinfection on the detection of Salmonella Enteritidis in shell eggs was also investigated. The results indicated that tryptic soy broth (TSB) was similar to TSB plus ferrous sulfate, but significantly (α = 0.05) better than nutrient broth, Universal Preenrichment broth, and buffered peptone water when used for preenrichment of Salmonella in shell eggs. Salmonella Enteritidis populations after enrichment with Rappaport-Vassiliadis broth were 0.40 to 1.11 log cfu/mL of culture lower than those in preenrichment cultures. The reduction was statistically significant (α = 0.05). Egg:broth ratios at 1:9 and 1:2 produced significantly (α = 0.05) higher Salmonella Enteritidis populations after preenrichment with TSB with inoculum levels at 4 cfu/100 g of eggs and 40 cfu/1,000 g of eggs than the ratio at 1:1. Salmonella Enteritidis populations in TSB preenrichment cultures of shell eggs surface-disinfected with 70% alcohol:iodine/potassium iodide solution and untreated control were 9.11 ± 0.11 and 9.18 ± 0.05 log cfu/mL, respectively, for SE 13-2, and 9.20 ± 0.04 and 9.16 ± 0.05 log cfu/mL, respectively, for SE CDC_2010K_1543. Surface disinfection of eggs did not reduce the sensitivity of detection of Salmonella Enteritidis in liquid eggs. These results could improve the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella in shell eggs by simplifying the preenrichment medium and changing the sample handling before enrichment.

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs.

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.

An improved method for the recovery of Salmonella serovars from orange juice using universal preenrichment broth.

The relative effectiveness of three methods for the recovery of Salmonella serovars from orange juice was determined. One method, a modified Bacteriological Analytical Manual (BAM) procedure consisted of preenrichment in lactose broth at 35 degrees C for 24 h, selective enrichment, and selective plating. Another method, a National Centers for Disease Control and Prevention (CDC 1) procedure, consisted of direct enrichment in tetrathionate broth at 35 degrees C for 24 and 48 h, followed by selective plating. The third method (also from CDC and designated CDC 2) consisted of preenrichment in Universal Preenrichment (UP) broth at 35 degrees C for 24 h, selective enrichment, and selective plating. In 10 experiments encompassing five different Salmonella serovars and 200 test portions per broth, the CDC 1 method recovered 141 Salmonella-positive test portions, the BAM method recovered 151, and the CDC 2 method recovered 171. In 2 of the 10 experiments, with two different Salmonella serovars, the BAM recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did the CDC 2 method. On the basis of the above results, the second phase of this study focused on a comparison of the effectiveness of the BAM-recommended lactose broth and the CDC 2-recommended UP broth as preenrichment media for the recovery of Salmonella serovars from pasteurized and unpasteurized orange juice. Subsequent culture treatment of the two preenrichments was identical so that the effect of other variables (e.g., different selective enrichment media, various incubation temperatures, and different selective plating agars) on the relative performance of these two preenrichment media was excluded. In one of nine experiments, with pasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the nine pasteurized orange juice experiments (180 test portions per broth), lactose broth recovered 99 Salmonella-positive test portions, and UP broth recovered 116. In three of seven experiments, with unpasteurized orange juice, lactose broth recovered significantly fewer (P < 0.05) Salmonella-positive test portions than did UP broth. For the combined results of the seven unpasteurized orange juice experiments (140 test portions per broth), lactose broth recovered 73 Salmonella-positive test portions, and UP broth recovered 117. For both pasteurized and unpasteurized orange juice, the total number of Salmonella-positive test portions recovered with UP broth was significantly greater than the number recovered with lactose broth. These results indicate that UP broth is a more effective enrichment broth for the recovery of Salmonella from orange juice than is lactose broth.

Rappaport-Vassiliadis medium for recovery of Salmonella spp. from low microbial load foods: collaborative study.

Twenty-three laboratories participated in a collaborative study to compare the relative effectiveness of Rappaport-Vassiliadis (RV) medium incubated at 42 degrees C, selenite cystine (SC) broth (35 degrees C), and tetrathionate (TT) broth (35 and 43 degrees C) for recovery of Salmonella from the following foods with a low microbial load: dried egg yolk, dry active yeast, ground black pepper, guar gum, and instant nonfat dry milk. For dry active yeast, lauryl tryptose (LT) broth, incubated at 35 degrees C, was used instead of SC broth. All of the foods were artificially inoculated with single Salmonella serovars, that had been lyophilized before inoculation, at high and low target levels of 0.4 and 0.04 colony forming units/g food, respectively. For analysis of 870 test portions, representing all of the foods except yeast, 249 Salmonella-positive test portions were detected by RV medium, 265 by TT broth (43 degrees C), 268 by TT broth (35 degrees C), and 269 by SC broth (35 degrees C). For analysis of 225 test portions of yeast, 79 Salmonella-positive test portions were detected by RV medium, 79 by TT broth (43 degrees C), 84 by TT broth (35 degrees C), and 68 by LT broth (35 degrees C). RV medium was comparable to, or even more effective than, the other selective enrichments for recovery of Salmonella from all of the foods except guar gum. It is recommended that RV (42 degrees C) and TT (35 degrees C) be used with foods that have a low microbial load, except for guar gum for which SC (35 degrees C) and TT (35 degrees C) are recommended.

Isolation of Salmonella spp. from the housefly, Musca domestica L., and the dump fly, Hydrotaea aenescens (Wiedemann) (Diptera: Muscidae), at caged-layer houses.

Flies, especially houseflies, are widely recognized as potential reservoirs and vectors of foodborne Salmonella pathogens. In this study, flies were collected at caged-layer facilities that had produced eggs that were implicated as the food vehicle in two recent outbreaks of Salmonella Enteritidis infections. The flies were separated by species into pools for microbiological testing. A total of 15 species pools of houseflies, Musca domestica L., and 7 species pools of bronze dump flies, Hydrotaea aenescens (Wiedemann) (Diptera: Muscidae), were analyzed. Salmonella Enteritidis was isolated from 2 of the 15 pools of houseflies. Other species of Salmonella were isolated from three pools of flies, including Salmonella Infantis from houseflies and from dump flies and Salmonella Heidelberg from houseflies. Salmonella Mbandaka was isolated from a lesser mealworm, Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae).

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load.

The relative effectiveness of Rappaport-Vassiliadis (RV) medium, selenite cystine (SC) broth, and tetrathionate (TT) broth for the recovery of Salmonella spp. from foods with a low microbial load was determined. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with a commercial preparation of SC broth, incubated at 35 degrees C, and TT broth incubated at 35 and 43 degrees C, for the recovery of Salmonella spp. Twenty-one artificially contaminated food types that included dairy foods, spices, and egg products, as well as other low-microbial-load foods, were analyzed. The foods were inoculated with single Salmonella serovars at target levels ranging from 0.04 to 0.4 CFU/g. No significant differences (P< or =0.05) among the selective enrichment broths for the recovery of Salmonella spp. from 18 of the foods were observed. Significantly fewer Salmonella-positive test portions of gelatin, guar gum, and nonfat dry milk were recovered with RV medium than with SC broth incubated at 35 degrees C and TT broth incubated at 35 and 43 degrees C. TT broth incubated at 35 degrees C recovered the greatest number of Salmonella-positive test portions. For the recovery of Salmonella spp. from foods with a low microbial load, it is recommended that TT broth incubated at 35 degrees C and RV medium incubated at 42 degrees C be used.

Methods for the recovery of Salmonella spp. from carboxymethylcellulose gum, gum ghatti, and gelatin.

Foods analyzed for Salmonella spp. by the procedure in the U.S. Food and Drug Administration's Bacteriological Analytical Manual are preenriched at a 1:9 test portion/broth ratio. Various thickening agents preenriched at this ratio become viscous and nonpipettable after 24 h incubation at 35 degrees C. The effects of 3 factors (presence of inorganic salts, adjustment of pH, and presence of enzymes) on the viscosity of the test portion/preenrichment mixtures of various thickening agents during incubation were determined. Reduction of the viscosities of these thickening agents was accomplished as follows: carboxymethylcellulose gum, addition of cellulase to a final concentration of 0.10% in lactose broth preenrichment and incubation with no pH adjustment; gum ghatti, addition of NaCl to a final concentration of 0.10% in lactose broth preerichment and adjustment of the pH to 6.5; and gelatin, addition of papain to a final concentration of 0.10% in lactose broth preenrichment and adjustment of the pH to 6.8. With these modified preenrichments, one Salmonella spp. cell/25 g (representing an approximate most probable number value of 0.04 cell/g) was generally recovered from the thickening agents.

Comparison of sorbitol MacConkey and hemorrhagic coli agars for recovery of Escherichia coli O157:H7 from brie, ice cream, and whole milk.

The relative efficacies of hemorrhagic coli (HC) agar and several formulations of sorbitol MacConkey (SorMac) agar, with and without 0.1% (w/v) 4-methyllumbelliferyl-beta-D-glucuronide (MUG), in recovering unstressed and heat-stressed Escherichia coli O157:H7 from Brie cheese, ice cream, and whole milk were determined. Recovery of unstressed E. coli O157:H7 was determined quantitatively by spread-plating diluted samples onto different agars and performing plate counts. Recovery of stressed E. coli O157:H7 was determined qualitatively by enriching samples in modified trypticase soy broth, streaking the incubated enrichments, and isolating E. coli O157:H7 colonies from the agars. HC agar and the SorMac agar formulations did not differ significantly in their ability to recover unstressed E. coli O157:H7 from ice cream and whole milk; however, HC agar recovered significantly more unstressed E. coli O157:H7 from Brie cheese than did the SorMac agar formulations. Bacteriological Analytical Manual and Oxoid SorMac agar formulations made from individual ingredients, did not differ significantly in recovering unstressed E. coli O157:H7 from Brie cheese. The efficiency of the commercially available Oxoid SorMac agar could not be determined because of overgrowth by indigenous microflora. HC and SorMac agars did not differ significantly in recovering stressed E. coli O157:H7 from Brie cheese, ice cream, and whole milk. MUG had no apparent effect on recovery of either stressed or unstressed E. coli O157:H7 from the dairy foods examined.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and rappaport-vassiliadis medium for recovery of Salmonella spp. from raw flesh, highly contaminated foods, and poultry feed: collaborative study.

A collaborative study was performed in 18 laboratories to validate use of Rappaport-Vassiliadis (RV) medium in the standard culture method for recovery of Salmonella spp. from raw, highly contaminated foods and poultry feed. RV medium made from its individual ingredients and incubated at 42 degrees C was compared with selenite cystine (SC) broth incubated at 35 degrees C and tetrathionate (TT) broth incubated at 35 degrees and 43 degrees C for effectiveness in recovery of Salmonella spp. Four artificially contaminated foods (oysters, frog legs, mushrooms, and shrimp) and poultry feed and one naturally contaminated food (chicken) were analyzed. The artificially contaminated foods were inoculated with single serovars of Salmonella at target levels of 0.04 colony-forming units (CFU)/g for the low level and 0.4 CFU/g for the high level. For analysis of 1125 test portions, RV medium (42 degrees C) recovered Salmonella from 409 test portions; TT (43 degrees C), from 368 test portions; TT (35 degrees C), from 310 test portions; and SC (35 degrees C), from 334 test portions. Overall, RV medium was comparable with or better than other selective enrichments for recovery of Salmonella from the foods in this study, except mushrooms. From mushrooms, SC broth (35 degrees C) recovered more positive test portions than did RV medium (42 degrees C) and TT broth (43 degrees C). The method for detection of Salmonella in raw, highly contaminated foods and poultry feed using RV medium has been adopted by AOAC INTERNATIONAL. AOAC Official Method 967.25, Salmonella in Foods, Preparation of Culture Media and Reagents, has been revised to include RV medium, and the applicability of AOAC Official Method 967.26, Salmonella in Foods, Detection, has been restricted to processed foods.

Usefulness of cellulase in recovery of Salmonella spp. from guar gum.

Most foods examined for Salmonella spp. by the procedure described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual are preenriched at a 1:9 sample/broth ratio. However, 25 g guar gum (an emulsifying agent) is not wetted completely in 225 mL of preenrichment broth, and after a 24 h incubation at 35 degrees C, the product is transformed into a viscous, nonpipettable mass. The effects of 4 factors (inorganic salts, pH, temperature, and various enzymes) on the viscosity of the sample/preenrichment mixture during incubation were determined. Addition of various inorganic salts or adjustment of pH from 4.0 to 9.0 had no significant effect on the viscosity of the incubated mixture. Elevated incubation temperatures of 42 degrees, 44 degrees, and 46 degrees C reduced viscosity but were well above the optimal growth temperature for Salmonella, 35 degrees C. Addition of cellulose to lactose broth at a final concentration of 0.01% reduced viscosity of the mixture, making it readily pipettable. At least one Salmonella cell was consistently recovered from 25 g samples of guar gum, which represents a most probable number value of 0.04 cell per g.

Relative effectiveness of selective plating agars for recovery of Salmonella species from selected high-moisture foods.

The relative effectiveness of 6 selective plating media were compared for effectiveness in recovery of Salmonella spp. from selected high-moisture foods. Three new plating agars (EF-18, Rambach, and xylose lysine Tergitol-4) and 3 selective plating agars (bismuth sulfite, Hektoen enteric, and xylose lysine desoxycholate) recommended by AOAC INTERNATIONAL and the Bacteriological Analytical Manual (BAM) were compared. The agars were streaked from cultures selectively enriched in selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium. The high-moisture foods studied were naturally contaminated pork sausage, chicken parts, turkey parts, and frog legs and artificially contaminated shrimp, oysters, egg yolks, and lettuce. The relative effectiveness of each selective plating agar was determined by recovery of Salmonella spp. and enumeration of false-positive and false-negative reactions. Although the new selective plating agars compared favorably with the AOAC/BAM-recommended agars, they offered no advantage. Incubation of selective enrichment broths at elevated temperatures decreased the numbers of false-positive and false-negative reactions for all 6 selective plating agars.

Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappaport-Vassiliadis medium for the recovery of Salmonella from raw flesh and other highly contaminated foods: precollaborative study.

The effectiveness of selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium for recovery of Salmonella spp. from 8 highly contaminated foods was determined. RV medium prepared from individual ingredients and incubated at 42 degrees and 43 degrees C was compared with 2 commercial (Difco and Oxoid) media incubated at 42 degrees C. Naturally and artificially contaminated foods were tested under 2 protocols. For Protocol 1, each food was preenriched in the appropriate medium. After incubation, serial 10 fold dilutions of the preenriched foods were inoculated into selective enrichment media and incubated at 35 degrees, 42 degrees, or 43 degrees C. Effectiveness of these conditions was evaluated by most probable number determination of Salmonella spp. recovered. Productivity of selective enrichments did not differ significantly with this protocol, except that with Oxoid RV medium the number of Salmonella cells recovered from most of the foods was significantly reduced. For Protocol 2, twenty 25 g test portions from artificially inoculated foods were examined qualitatively for Salmonella spp. The effectiveness of the broth/temperature combinations was determined by the number of positive tests under each condition. RV medium prepared from individual ingredients and TT broth incubated at 43 degrees C yielded significantly more Salmonella-positive tests from frog legs and lettuce than did SC and TT broths incubated at 35 degrees C or commercial RV medium incubated at 42 degrees C. With pork sausage and ground beef, significantly fewer Salmonella-positive tests were found with Oxoid RV medium incubated at 42 degrees C and SC incubated at 35 degrees C than from other selective enrichments.(ABSTRACT TRUNCATED AT 250 WORDS)

Effectiveness of a one-day procedure for recovery of Salmonella from milk powders.

A rapid procedure for enumerating Salmonella in milk powders was evaluated. Dry whole milk and instant nonfat dry milk were rehydrated, artificially inoculated with various numbers of Salmonella cells, and stomached. Test portions were then treated with Tween 80 and pancreatic trypsin, and incubated for 1 h at 30 degrees C. The incubated test portions were centrifuged at 10,000 x g for 15 min at 5 degrees C, and the resuspended pellets were plated on xylose lysine desoxycholate agar. The effectiveness of the procedure was expressed in terms of percentage recovery of the inoculum. The procedure, which was evaluated in 76 trials using 7 Salmonella serovars, recovered < or = 73% of the inoculum for half of the trials conducted. Its effectiveness was dependent on the serovar, level of inoculation, and type of milk powder used.

Effectiveness of the Bacteriological Analytical Manual culture method for the recovery of Shigella sonnei from selected foods.

The relative retention of the indigenous morphological, biochemical, and serological characteristics by Shigella sonnei was tested under various storage conditions (room temperature, refrigeration, freezing at -20 degrees C and at -70 degrees C, and lyophilization). The use of a selective (desoxycholate citrate) agar rather than a nonselective (brain heart infusion) agar gave a lower conversion rate of smooth to rough colonies, and the percentage of rough colonies derived from cultures stored for prolonged periods increased under all conditions. With respect to biochemical characteristics, there were no major differences in the reactions of smooth vs rough variants. For serological characteristics, smooth variants agglutinated more readily in homologous antisera than did rough variants. S. sonnei populations maintained at -70 degrees C with glycerol remained reasonably stable and were used in recovery studies. Up to six foods (potato salad, chicken salad, cooked salad shrimp, lettuce, raw ground beef, and raw oysters) were inoculated with unstressed, chill-stressed, or freeze-stressed S. sonnei cells. Test portions (25 g) were inoculated with serial 10-fold dilutions of culture and subsequently analyzed by the culture method described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual. It was found that the method was relatively ineffective for the recovery of S. sonnei from raw ground beef and raw oysters.

Research note: growth of Salmonella enteritidis in Grade A eggs during prolonged storage.

Migration of Salmonella enteritidis through egg albumen to the yolk and its subsequent growth in the yolk were examined. Submersion of eggs in .1% mercuric chloride solution for 1 h followed by submersion in 70% ethanol for 30 min resulted in an eggshell surface from which no Salmonella organisms were recovered. The eggs were then inoculated with S. enteritidis under the shell membrane. Although growth of S. enteritidis was negligible in eggs refrigerated up to 16 days, the population level of the organism increased by more than 8 log10 units in unrefrigerated eggs stored for the same amount of time.