Rapid Determination of Salmonella in Samples of Egg Noodles, Cake Mixes, and Candies

A glass apparatus system was compared with a standard enrichment broth-selective agar method to test samples of egg noodles, cake mixes, and candy for the presence or absence of salmonellae. The glass apparatus system used fermentation of mannitol, production of H(2)S, or motility, in conjunction with a serological test of flagellar antigens, to detect salmonellae. No salmonellae were detected in 173 samples of food products. Of these samples, 171 were found to be Salmonella-negative after 48 hr with the glass apparatus system. After 72 hr, the standard Salmonella procedure yielded 38 samples which produced Salmonella false-positive results on selective agars. Inoculation of samples with cultures of Salmonella showed that approximately one inoculated cell could be detected after 48 hr of incubation with the glass apparatus. The standard Salmonella test requires a minimum of 72 hr for completion. Compared with the standard Salmonella test, the glass apparatus system is a more rapid and simple system that can be used to determine the presence or absence of Salmonella in these food products.     

Evaluation of an automated fluorescent antibody procedure for detection of Salmonella in foods and feeds.

A prototype automated system using fluorescent antibody (FA) was evaluated for rapid detection of salmonellae in foods. Samples were enriched in selenite cystine and tetrathionate broths. After incubation, both were transferred into fresh selenite cystine for a 4-h "post-enrichment" to dilute possible background fluorescence from product. These cultures were then analyzed automatically, and results were compared with those obtained by the methods of the Association of Official Analytical Chemists (AOAC). Initially, 167 samples of milk powder, dried yeast, and imported frog legs were examined. The AOAC and automated FA methods correlated well with all samples but frog legs. Difficulty with the latter was caused by procedural and mechanical problems coupled with high numbers of competing microorganisms in post-enrichment cultures. Modification of procedure and partial redesign of equipment corrected these difficulties, and excellent correlation was obtained with another 116 frog leg samples. All 89 AOAC-confirmed positives were also detected by the automated FA method, and there were only 4% false FA positives. The system shows potential for screening products for salmonellae; however, all positives should be confirmed by manual biochemical and serological methods.     

Rapid, Direct Fluorescent-Antibody Method for the Detection of Salmonellae in Food and Feeds

An improved immunofluorescent-antibody (FA) method for the detection of salmonellae in foods and feeds was developed. This FA method combines a rapid cultural phase and a serological phase that allow for propagation of salmonellae in a minimum time, employing the industrial 8-hr work day as a guide. Two hundred fifty naturally contaminated human food and animal feed samples, representing 647 trials, were tested by the FA method. A total of 18 different food and feed samples was used. The method used by the Association of Official Analytical Chemists (AOAC) for the detection of salmonellae was the control method. The percent agreement when comparing the FA slide method to the AOAC method ranged from 87.1 to 95.3%, depending upon the conjugated antisera used in comparative studies.     

Further Evaluation of a Rapid Immunofluorescence Technique for Detecting Salmonellae in Meat and Poultry

A rapid 18–24 h immunofluorescence technique detected 14 of 15 positive samples in tests on 706 routine samples, which included 656 home produced raw beef samples. The rapid technique also recorded 49 false positive results, i.e. samples which proved negative in subsequent cultural tests. The immunofluorescence technique could be used as a presumptive screening test aimed at the rapid detection of negative samples. In this way salmonella free raw materials should usually be cleared for production within 1 day of sampling.     

Comparison of Two Procedures for Detection of Salmonella in Food, Feed, and Pharmaceutical Products

A survey of food, feed, and pharmaceutical products was undertaken to compare an accelerated Salmonella detection procedure by enrichment serology (ES) to the traditional procedure outlined in the Bacteriological Analytical Manual (BAM). Excellent agreement between the two methods was obtained in the results of 689 test samples involving 35 different products. In addition to being more rapid and simpler to perform, the ES procedure was just as accurate and sensitive as the BAM procedure.     

The Use of Enrichment Serology for Salmonella Detection in Human Foods and Animal Feeds

S ummary . Samples (2208) of food raw materials and products were examined for the presence of salmonellae by use of conventional salmonella detection procedures and the enrichment serology (ES) techniques described by Sperber & Deibel (1969); 348 samples were positive for salmonellae by the conventional procedures. Using the ES technique with a 24 h elective enrichment step, 93–98% of samples positive by the conventional procedures were also positive by the ES technique. Selective enrichment of food samples using tetrathionate broth containing novobiocin, incubated at 41°, led to the best recovery of salmonellae by both the conventional and ES techniques.     

A Rapid, Presumptive Procedure for the Detection of Salmonella in Foods and Food Ingredients

A rapid detection procedure was developed in which a lysine-iron-cystine-neutral red (LICNR) broth medium, originally described by Hargrove et al. in 1971, was modified and used to detect the presence of viable Salmonella organisms in a variety of foods, food ingredients, and feed materials by using a two-step enrichment technique. Tetrathionate broth was used to enrich samples with incubation at 41 C for 20 hr, followed by transfer to LICNR broth and incubation at 37 C for 24 hr for further enrichment and for the detection of Salmonella organisms by color change. One hundred ten samples representing 18 different sample types were evaluated for the presence of viable Salmonella. Ninety-four percent of the samples found to be presumptive positive by this method were confirmed as positive by a culture method. Fluorescent-antibody results also compared closely. A second study was conducted under quality-control laboratory conditions by using procedures currently employed for Salmonella detection. One hundred forty-three samples representing 19 different sample types were evaluated for the presence of viable Salmonella. No false negatives were observed with the rapid-detection method. The usefulness of the LICNR broth procedure as a screening technique to eliminate negative samples rapidly and to identify presumptive positive samples for the presence of viable Salmonella organisms was established in this laboratory.     

Rapid Detection of Salmonella Microcolonies by Fluorescent Antibody

A microcolony fluorescent-antibody (FA) procedure for detecting salmonellae was compared to the usual direct FA procedure on 304 environmental, food, and feed samples. The microcolony FA test detected all of the specimens found positive by culture, whereas the direct FA missed 3.1% of them. Both FA tests revealed stained organisms in some of the culturally negative specimens. The microcolony FA test has several advantages over the direct FA test: ease of examining the smears, elimination of the fluorescent background material, and increased sensitivity.     

Fluorescent-Antibody Technique in Detection of Salmonellae in Animal Feed and Feed Ingredients

Comparative studies of fluorescent antibody procedure and a cultural method for the detection of Salmonella were made on 1,013 feed and feed-ingredient samples. The agreement between the two methods was 92.1%. There were more false positives (5.7%) than false negatives (2.2%). Of the 22 false negatives, 15 (68%) were obtained on meat meal. Of the total number of samples, 37% were meat meal. An additional study of 73 samples of meat meal indicated that correlation between methods was better than correlation between samples.     

Comparison of membrane filter, multiple-fermentation-tube, and presence-absence techniques for detecting total coliforms in small community water systems

Methods for detecting total coliform bacteria in drinking water were compared using 1,483 different drinking water samples from 15 small community water systems in Vermont and New Hampshire. The methods included the membrane filter (MF) technique, a 10-tube fermentation tube (FT) technique, and the presence-absence (P-A) test. Each technique was evaluated using a 100-ml drinking water sample. Of the 1,483 samples tested, 336 (23%) contained coliforms as indicated by either one, two, or all three techniques. The FT detected 82%, the P-A detected 88%, and the MF detected 64% of these positives. All techniques simultaneously detected 55% of the positives. Evaluation of the confirmation efficiency of the P-A technique showed 94% of the presumptive positives confirming as coliforms. Thirteen different species of coliforms were identified from the 37 tests in which the P-A was positive but the MF and FT were negative. The P-A test was simple to inoculate and interpret and was considerably more sensitive than the MF and slightly more sensitive than the FT in detecting coliforms in this type of drinking water supply.     

Comparison of Fluorescent-Antibody Methods and Enrichment Serology for the Detection of Salmonella

Four rapid methods for detection of Salmonella, (i) the conventional fluorescent-antibody (FA) technique, (ii) a rapid direct FA technique, (iii) microcolony FA, and (iv) enrichment serology (ES), were compared with conventional cultural procedures. A total of 347 subsamples representing 16 different food prototypes, alleged to be naturally contaminated with Salmonella, were analyzed. From these samples, 52 were found to contain Salmonella by cultural methods. Conventional FA identified all 52 culturally positive samples, ES identified 51, microcolony FA identified 48, and the rapid FA method identified 34. The number of false-positive samples for each procedure was: ES-selenite, 7; tetrathionate, 8; rapid FA, 26; microcolony FA, 33; conventional FA-selenite, 27; tetrathionate, 26. Tetrathionate enrichment was found to be superior to selenite for Salmonella recovery from most foods, but the concurrent use of both media allowed maximum recovery.     

Preparation and Testing of Polyvalent Conjugates for Fluorescent-Antibody Detection of Salmonellae

A polyvalent OH conjugate for Salmonella O groups A through I, K, L, and O was prepared and tested against pure cultures of salmonellae, nonsalmonellae, and a variety of food, fecal, and environmental specimens. Examination of pure cultures revealed that the conjugate gave negligible staining with representative strains of Shigella, Proteus, Providence, Serratia, and Pseudomonas. However, it stained 12% of the Escherichia coli and Citrobacter freundii strains and 36% of the Arizona strains. Over 1,200 specimens of various types were examined by both fluorescent-antibody (FA) and cultural procedures. Results indicate that, when used with discretion, FA screening can be a useful tool for rapid presumptive indication of the presence of salmonellae. The need for careful selection of strains used for preparing antisera and the importance of adequate evaluation of Salmonella FA reagents are discussed.     

Effect of Type of Enrichment and Duration of Incubation on Salmonella Recovery from Meat-and-Bone Meal

Twenty-five meat-and-bone meal samples were enriched with either selenite-cystine or tetrathionate and incubated for 1 and 2 days. Seven were previously found to be positive; of the other 18, 16 were positive for salmonella. The number of somatic serogroups per sample ranged from 1 to 11 with a mean of 3.8. Significantly more (P < 0.01) group C₁ salmonellae were isolated using tetrathionate than selenite, whereas significantly more of groups G, 35, and Difco poly-valent D were isolated from selenite than tetrathionate. Seventy-six percent of the presumptive colonies from Brilliant Green agar showed a positive lysine decarboxylase reaction, and there were no differences between media or times of incubation. Ninety-four per cent of the lysine decarboxylase-positive cultures showed a positive somatic antiserum response; again there were no differences between times or enrichments although there were significantly more total positive serogroups at 2 days than at 1 day from tetrathionate but not from selenite. There were indications that certain serogroups preferred either one or the other enrichment. There were no differences in total positive samples with the two enrichments although neither alone was sufficient to identify all positives. Several lactose-positive salmonellae were recovered.     

Identification of Salmonella with the O-1 Bacteriophage

The O-1 bacteriophage test of Cherry et al. (1954) for the presumptive identification of salmonellae in the diagnostic laboratory was investigated. A phage lysate with a titer of 10(12) plaque-forming units per ml was found to be optimal. This preparation lysed 98.2% of Salmonella strains tested, while maintaining its high specificity for salmonellae. Gram-negative organisms other than salmonellae were resistant to the O-1 phage; however, 5.9% of Escherichia coli strains tested were susceptible. The O-1 phage test is a simple, rapid, inexpensive, sensitive, and specific procedure for the identification of salmonellae in the diagnostic laboratory. A presumptive identification is obtained 1 day earlier than with conventional biochemical tests.     

Improvement of the detection of Listeria monocytogenes by the application of ALOA, a diagnostic, chromogenic isolation medium

A new selective agar medium, ALOA, for the selective and differential isolation of Listeria monocytogenes has been evaluated. All stressed cultures of L. monocytogenes serovars tested grew on the medium as bluish colonies surrounded by a distinctive opaque halo and gave a productivity ratio of at least 0.95. Non-pathogenic Listeria sp. produced bluish colonies without a halo as was also the case for some enterococci and bacilli. Special attention must be paid to some Bacillus cereus strains and L. ivanovii since their colony appearance can be misleading. Only some unidentified listeria-like bacteria gave false-positive results. ALOA detected 4. 3% more positives from naturally contaminated dairy and meat samples compared with the ISO procedure when used with GenprobeTM or VidasTM for confirmation of presumptive colonies; 13.9% false negatives were found compared with 38.9% using PALCAM/Oxford. ALOA was also clearly superior to Oxford and PALCAM when samples containing both L. monocytogenes and L. innocua were examined. The introduction of ALOA in standard isolation procedures as an additional medium would enhance the detection ratio and reduce the time and cost of analysis for L. monocytogenes.     

Direct Fluorescent-Antibody Technique for the Microbiological Examination of Food and Environmental Swab Samples for Salmonellae

Comparative studies of a modified fluorescent-antibody procedure and the 5 to 7 day method used by the Association of Official Analytical Chemists for the detection of Salmonella were made on 151 samples of wheat products and 183 swab samples. The agreement between the two methods for the 334 samples tested was 92.5%. Food samples yielded 94.7% agreement, whereas the swab samples yielded 90.7% agreement. There were 7.5% false positives for the total number of samples tested. No false negatives were obtained by using the fluorescent-antibody method.     

Enchanced accuracy of coliform testing in seawater by a modification of the most-probable-number method.

A 1-year study of marine water sample from six beach locations showed that the most-probable-number method failed to recover significant numbers of coli-forms. Modifying this method by transferring, after 48 h, presumptive negatives (growth and no gas production) to confirmed and fecal coliform media significantly improved recovery. Tests which were presumptive negative but confirmed as fecal coliform positive were designated as false negatives. Most-probable-number method false negatives occurred throughout the year, with 143 of 270 samples collected producing false negatives. More than 50% of fecal coliform false-negative isolates were Escherichia coli. Inclusion of false-negative tubes into the coliform most-probable-number method data resulted in increased violation of the California ocean water contact sports standard at all sites. More than 20% of the samples collected were in violation of this standard. These data indicate that modification of the most-probable-number method increases detection of coliform numbers in the marine environment.     

Enchanced accuracy of coliform testing in seawater by a modification of the most-probable-number method.

A 1-year study of marine water sample from six beach locations showed that the most-probable-number method failed to recover significant numbers of coli-forms. Modifying this method by transferring, after 48 h, presumptive negatives (growth and no gas production) to confirmed and fecal coliform media significantly improved recovery. Tests which were presumptive negative but confirmed as fecal coliform positive were designated as false negatives. Most-probable-number method false negatives occurred throughout the year, with 143 of 270 samples collected producing false negatives. More than 50% of fecal coliform false-negative isolates were Escherichia coli. Inclusion of false-negative tubes into the coliform most-probable-number method data resulted in increased violation of the California ocean water contact sports standard at all sites. More than 20% of the samples collected were in violation of this standard. These data indicate that modification of the most-probable-number method increases detection of coliform numbers in the marine environment.     

Comparison of membrane filter, multiple-fermentation-tube, and presence-absence techniques for detecting total coliforms in small community water systems.

Methods for detecting total coliform bacteria in drinking water were compared using 1,483 different drinking water samples from 15 small community water systems in Vermont and New Hampshire. The methods included the membrane filter (MF) technique, a 10-tube fermentation tube (FT) technique, and the presence-absence (P-A) test. Each technique was evaluated using a 100-ml drinking water sample. Of the 1,483 samples tested, 336 (23%) contained coliforms as indicated by either one, two, or all three techniques. The FT detected 82%, the P-A detected 88%, and the MF detected 64% of these positives. All techniques simultaneously detected 55% of the positives. Evaluation of the confirmation efficiency of the P-A technique showed 94% of the presumptive positives confirming as coliforms. Thirteen different species of coliforms were identified from the 37 tests in which the P-A was positive but the MF and FT were negative. The P-A test was simple to inoculate and interpret and was considerably more sensitive than the MF and slightly more sensitive than the FT in detecting coliforms in this type of drinking water supply.     

Efficiency of Salmonella Isolation from Meat-and-Bone Meal of One 300-g Sample Versus Ten 30-g Samples

Twenty-five meat-and-bone meal samples were analyzed for salmonellae, comparing a single 300-g to ten 30-g samples. Seventeen were positive using the larger sample; eighteen were positive with the smaller. The 300-g sample showed a significantly higher (P < 0.01) percentage of confirmed salmonellae at 2 days of incubation than at 1 day. The ten 30-g samples did not show changes at 2 days. At 2 days, the 30-g samples showed significantly fewer confirmed salmonellae than the 300-g sample; however, there was no difference at 1 day. Of 1,417 presumptive colonies picked, 1,215 (85.7%) were lysine decarboxylase-positive and 1,152 (81.3%) were agglutinated by one of the somatic antisera. There were no significant differences in diversity or total numbers of different somatic groups between the large and small samples.