Twenty-Four-Hour Immunofluorescence Technique for the Detection of Salmonellae in Nonfat Dry Milk

A detection procedure was developed in which a newly devised lysine-iron medium was used as a one-step selective and enrichment medium for detection of salmonellae by the fluorescent-antibody technique. Incubation was conducted in two steps: initially at 30 C for 5 hr to resuscitate sublethally stressed cells, followed by incubation at 39 C for 17 hr. Twenty-seven strains of salmonellae from groups A-I were utilized in the development of this procedure which was sensitive enough to detect one Salmonella bacterium in 100 g of nonfat dry milk.     

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.     

A membrane-immunofluorescent-viability staining technique for the detection of Salmonella spp. from fresh and processed meat samples

A direct staining technique was investigated for the detection of viable Salmonella in fresh and processed meats. The technique involved overnight enrichment in BPW, extraction of Salmonella cells onto a polycarbonate membrane, followed by detection of the pathogen using anti-Salmonella monoclonal antibody coupled with an antibody linked-fluorescent stain (Texas Red) and a viability stain (Sytox Green). The technique was applied to the detection of Salm. enteritidis inoculated into broth culture or minced beef and then subjected to a variety of stresses including freezing (- 20 degrees C), heating (2 or 4 min at 56.9 degrees C), low pH (5 or 3.5) or high salt (2 or 4%). The correlation between traditional plate counts and the rapid count varied widely (r2 = 0.98-0.03), depending on the type and level of stress applied to the cells. The reason for the disparity in results obtained, and the potential application of the method as a diagnostic tool, are discussed.     

Rapid Fluorescent-Antibody Staining Technique

The rapid fluorescent-antibody staining technique described by Kellogg and Deacon for staining Neisseria gonorrhoeae and Treponema pallidum was applied to fluorescent-antibody tests for group A streptococci and enteropathogenic Escherichia coli. Results obtained with this staining technique were compared with results using the conventional staining procedure; excellent correlation was obtained. Considerable time and materials were saved by using the rapid method; it was also found completely satisfactory.     

Evaluation of the Vitek Immunodiagnostic Assay System (VIDAS) for the detection of Salmonella in foods

A Salmonella Assay using the Vitek Immunodiagnostic Assay System (VIDAS) was compared with a conventional cultural method (CCM) for the detection of salmonellas in 141 samples of artificially and naturally contaminated foods. There was an overall agreement of 92.9% between the methods. The productivity of the VIDAS Salmonella Assay (VSA) was not improved using an alternative enrichment protocol for the detection of Salmonella in 12 raw meat samples.
The sensitivity and specificity of the VSA was assessed using pure cultures of salmonellas and non-salmonellas. The detection limit was 1.8 times 10⁶ salmonellas ml^-1 in M-broth and some Citrobacter freundii strains gave false-positive results.
Using an immunomagnetic separation (IMS) technique and an abbreviated cultural enrichment, the VSA results could be obtained a day earlier than the standard VSA method.     

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.     

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.     

Detection of Chlamydia psittaci by Immunofluorescence

A direct fluorescent-antibody (FA) test was developed to detect Chlamydia psittaci in dural impressions from specimen-inoculated mice. Technical procedures for the test were compared. C. psittaci was found in mice after infection as early by the FA technique as it was by cytochemical staining methods usually used. The lymphogranuloma venereum organism was also stained by conjugated antibody to C. psittaci. A distinctive advantage of the described FA test is that organisms are identified immunologically as members of the genus Chlamydia simultaneously with their detection.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality.     

Agar Block Technique for Identification of Mycoplasmas by Use of Fluorescent Antibody

A procedure for staining mycoplasmata colonies directly on agar blocks for examination by fluorescent microscopy is described. Areas of the agar surface appropriate for staining were demarcated by use of Lucite cylinders. Direct fluorescent-antibody staining was superior to indirect staining. The technique was very useful for determining whether cultures were mixed and for identification of mycoplasmas in either pure or mixed cultures.     

Typing Herpesvirus hominis Antibodies and Isolates by Inhibition of the Indirect Hemagglutination Reaction

Inhibition of the indirect hemagglutination reaction (IHA inhibition) was compared to several other methods for type-specific identification of Herpesvirus hominis (HVH) antibodies and isolates. The method appears to have the greatest value for typing antibodies for HVH type 1 and HVH type 2 in human sera; identification of antibody type was relatively simple and results were definitive. The IHA-inhibition test permitted serological diagnosis of HVH type 2 infection in three young adults with meningoencephalitis, thus extending the mounting evidence that nervous system involvement with this virus type is not limited to neonatal infections. II/I indexes of neutralizing or IHA antibody gave an accurate indication of the presence of HVH type 2 antibody in those sera containing type 2 antibody by IHA inhibition, but they indicated the presence of HVH type 2 antibody in one-half or more of the sera shown to contain only HVH type 1 antibody by IHA inhibition. For typing HVH isolates, the IHA-inhibition test gave results identical to those obtained by direct fluorescent-antibody staining using cross-absorbed conjugates, but the IHA-inhibition test was much more cumbersome and time-consuming to perform than was direct fluorescent-antibody staining. A microneutralization technique for virus typing also gave results identical to those obtained with direct fluorescent-antibody staining and IHA inhibition. However, typing HVH isolates by plaque size or the differential effect of incubation temperature was found to be less definitive and accurate.     

Use of a Rapid Fluorescent-Antibody Staining Technique with Group A Streptococci

A pilot study concerning a rapid fluorescent-antibody staining technique for identification of group A streptococci is described. Results indicate the method merits further study.     

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.     

Use of enzyme-labeled antibodies to detect Salmonella in foods.

An indirect enzyme-labeled antibody technique (ELAT), in which Salmonella typhimurium was used as a model, was developed as a method to detect Salmonella in food samples. A cellulose-acetate membrane filter, the matrix for detection, was placed on a membrane-filter base and overlaid with a multiwelled lucite template. Mixed broth enrichment cultures were dispensed in the template wells, and cells were spotted onto the membrane via suction. After fixation, the membranes were immersed in rabbit anti-S. typhimurium flagella antibody, washed, immersed in goat anti-rabbit antibody conjugated to peroxidase, and washed. Exposure of membranes to the substrates 3,3'-diaminobenzidine or benzidine resulted in development of brown or blue macroscopic reaction products, respectively, on spots containing S. typhimurium. ELAT results agreed with those of enrichment serology and cultural procedures on three food products containing known levels of S. typhimurium. Because of the magnification effect of the enzyme-substrate reaction, fewer cells were needed for detection than with enrichment serology, thereby reducing the total analysis time. The ability to test 14 or more samples simultaneously on a 47-mm membrane filter would facilitate screening large number of samples. Pending the development of a pure H antisera pool for the common Salmonella serotypes free from O antibodies, the ELAT demonstrated potential as a Salmonella detection methodology.     

Use of enzyme-labeled antibodies to detect Salmonella in foods.

An indirect enzyme-labeled antibody technique (ELAT), in which Salmonella typhimurium was used as a model, was developed as a method to detect Salmonella in food samples. A cellulose-acetate membrane filter, the matrix for detection, was placed on a membrane-filter base and overlaid with a multiwelled lucite template. Mixed broth enrichment cultures were dispensed in the template wells, and cells were spotted onto the membrane via suction. After fixation, the membranes were immersed in rabbit anti-S. typhimurium flagella antibody, washed, immersed in goat anti-rabbit antibody conjugated to peroxidase, and washed. Exposure of membranes to the substrates 3,3'-diaminobenzidine or benzidine resulted in development of brown or blue macroscopic reaction products, respectively, on spots containing S. typhimurium. ELAT results agreed with those of enrichment serology and cultural procedures on three food products containing known levels of S. typhimurium. Because of the magnification effect of the enzyme-substrate reaction, fewer cells were needed for detection than with enrichment serology, thereby reducing the total analysis time. The ability to test 14 or more samples simultaneously on a 47-mm membrane filter would facilitate screening large number of samples. Pending the development of a pure H antisera pool for the common Salmonella serotypes free from O antibodies, the ELAT demonstrated potential as a Salmonella detection methodology.     

Legionella contamination of dental-unit waters.

Water samples collected from 28 dental facilities in six U.S. states were examined for the presence of Legionella pneumophila and other Legionella spp. by the PCR-gene probe, fluorescent-antibody microscopic, and viable-plate-count detection methods. The PCR and fluorescent-antibody detection methods, which detect both viable and viable nonculturable Legionella spp., gave higher counts and rates of detection than the plate count method. By the PCR-gene probe detection method, Legionella spp. were detected in 68% of the dental-unit water samples and L. pneumophila was detected in 8%. Concentrations of Legionella spp. in dental-unit water reached 1,000 organisms per ml or more in 36% of the samples, and 19% of the samples were in the category of 10,000/ml or above. L. pneumophila, when present in dental-unit water, never reached concentrations of 1,000/ml or more. Microscopic examination with fluorescent-antibody staining indicated that the contamination was in the dental-unit water lines rather than in the handpieces. Legionella spp. were present in 61% of potable water samples collected for comparative analysis from domestic and institutional faucets and drinking fountains; this percentage was not significantly different from the rate of detection of Legionella spp. in dental-unit water. However, in only 4% of the potable water samples did Legionella spp. reach concentrations of 1,000 organisms per ml, and none was in the 10,000 organisms-per-ml category, and so health-threatening levels of Legionella spp. in potable water were significantly lower than in dental-unit water. L. pneumophila was found in 2% of the potable water samples, but only at the lowest detectable level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Viral Identification by Scanning Electron Microscopy of Preparations Stained with Fluorescein-Labeled Antibody

A new technique combines the specificity of fluorescent-antibody labeling and the resolution of the scanning electron microscope to identify and distinguish between viruses. Hemagglutination of chicken erythrocytes by influenza virus was used as a model system to demonstrate the technique.     

Comparison of methods for isolating Salmonella bacteria from faeces of naturally infected pigs

A series of experiments was conducted using faecal samples collected from commercial swine farms to evaluate the effects of variation in methods used for the detection of Salmonella bacteria. The primary objective of the studies was to compare the protocols routinely used in two laboratories in the USA. The studies included five experiments comparing the enrichment protocols used routinely in the respective laboratories (Method 1: 10 g faeces--buffered peptone water (BPW) pre-enrichment--selective enrichment in Rappaport/Vassiliadis (RV) broth; Method 2: approximately 1g faeces--primary enrichments in tetrathionate and Hajna GN broths--secondary enrichment in RV broth). The effects of enrichment temperatures (37 vs 42 degrees C) using RV broth (two experiments) and delayed secondary enrichment (four experiments) were also evaluated. Direct comparison of Method 1 and Method 2 indicated comparable results. However, when compared using faecal samples of equal weight, the Method 2 enrichment protocol was more sensitive for detecting Salmonella bacteria than the Method 1 protocol. Enrichment in RV at 42 degrees C was superior to 37 degrees C, particularly for samples that were pre-enriched in BPW. Delayed secondary enrichment increased detection of Salmonella bacteria in swine faeces. These results highlight the imperfect sensitivity of culture methods, and the need for researchers to consider the sensitivity of bacteriological methods in the design and interpretation of the results of epidemiologic studies based on faecal culture.     

Identification ofEscherichia coli spheroplasts by the fluorescent-antibody staining technique

Spheroplasts ofEscherichia coli were produced by penicillin or lysozyme-ethylenediaminetetraacetic acid and examined by the direct fluorescent-antibody staining technique. Most spheroplasts stained with somatic-O fluorescent antibody exhibited brilliant peripheral fluorescence with localized areas of irregular staining. Electron micrographs showed that these spherical structures had considerable amounts of cell wall fragments associated with them. Two strains ofE. coli employed in the present study required different concentrations of penicillin for the conversion of all cells in an exponential culture to spheroplasts. Slight differences in lysozyme sensitivity were also encountered with these strains. The direct fluorescent-antibody staining technique was effective for the rapid identification ofE. coli spheroplasts in mixed cultures.