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.     

Detection of Salmonella in Eggs and Egg Products With Fluorescent Antibody

Organisms of the genus Salmonella are detected in eggs and egg products within 24 hr in the presence of Pseudomonadaceae and other Enterobacteriaceae by combining selective cultural methods with fluorescent-antibody techniques. These techniques are specific for Salmonella when H antibodies are used. Absorption techniques are necessary before the O antibodies give specific reactions for Salmonella. No cross-reactions appear when H antiserum is used. Absorption and interference techniques indicate the test is specific for Salmonella.     

Detection of Air-borne Pasteurella tularensis Using the Fluorescent Antibody Technique

Considerable research has been directed toward the development of rapid methods for the identification of air-borne microorganisms. The application of the fluorescent antibody technique (FAT) coupled with the impaction of contaminated air onto glass slides affords a rapid and specific method for the identification of air-borne Pasteurella tularensis. Early experiments presented problems of cross-reaction with organisms other than P. tularensis. These cross-reactions are eliminated by specific adsorption and proper dilution of the conjugate. A series of experiments conducted under rigidly controlled laboratory conditions indicates that fewer than ten viable P. tularensis per slide can be detected by this method. Time of impaction as well as the presence of large concentrations of other microorganisms did not alter this number. Calculations indicate that a concentration as low as one viable organism per 5 liters of air can be detected.     

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.     

Use of Gelatinase in the Routine Sampling of Gelatin for Salmonella Contamination

Gelatinase added to gelatin enrichment broth suspensions decreased viscosity. Salmonellae were not harmed by enzyme. Salmonella isolation procedures can be simplified by eliminating the viscosity problem.     

Evaluation of Commercial Conjugates for Fluorescent Antibody Detection of Salmonellae

Fluorescent antibody (FA) reagents for Salmonella produced by Difco, Sylvana, and Clinical Sciences, Inc., were evaluated for physicochemical and performance characteristics. The Difco panvalent (A through 064) and the Difco polyvalent (A through S) were similar in physicochemical characteristics. They had less than 60% gamma globulin with 3% albumin and had fluorescein to protein (F/P) ratios of less than 10. The Sylvana conjugate had 81% gamma globulin with less than 1% albumin. Its F/P was 33.9. The Clinical Sciences reagent contained 75% unlabeled albumin as packaged in the Fluoro-kit. Analysis of the original conjugate showed 86.5% gamma globulin with only 0.5% albumin. The (F/P) was 32.8. The performance characteristics were determined by using a variety of Enterobacteriaceae and food and feed samples. All conjugates stained the homologous Salmonella strains. The majority of cross-reactions were limited primarily to the Arizona, Citrobacter, and Escherichia coli groups. The Difco panvalent was more reactive with heterologous organisms. It stained 89% of the Arizona compared with 42% stained by the Difco polyvalent (A through S) and 39% stained by the Sylvana and Clinical Sciences reagents. We found 90% agreement between FA and culture when the Difco polyvalent was used to examine food and feed samples and 94% agreement when the Clinical Sciences Fluoro-kit was used on another group of samples.     

Evaluation of the Interference Filter for Use in Rabies Diagnosis by the Fluorescent Antibody Test

When compared with primary filters widely used for rabies diagnosis by the fluorescent antibody test, an interference filter markedly increased specific staining intensity and contrast.     

Evaluation of Frozen Fixed Smears for Use in Fluorescent Antibody Studies of Salmonellae

Smears of broth cultures of 28 Salmonella serotypes were fixed with Kirkpatrick fixative and stored at -20 C. Results indicate that organisms retain the ability to stain at maximal fluorescence intensity for as long as 2 years.     

The Use of an Improved Fluorescent Antibody Procedure in the Demonstration of Leptospira in Animal Tissues

Increased sensitivity of the fluorescent antibody procedure was achieved by modification of the method employed. Specimens collected from experimentally infected laboratory animals and naturally infected domestic and wild animals were examined by means of this method. The results compared very favorably with those obtained by other investigators who used serological, cultural, animal inoculation and silver impregnation procedures on the same specimens.     

Evaluation of a Semiautomated System for Direct Fluorescent Antibody Detection of Salmonellae

A semi-automatic system under development by Aerojet Medical and Biological Systems for the direct fluorescent antibody detection of salmonellae was evaluated with various food, feed, and environmental samples. All samples were simultaneously examined by Automated Bioassay System (ABS), manual direct fluorescent antibody procedures and cultural procedures. The ABS gave satisfactory results with the processed samples. It detected all of the culturally positive powdered egg and candy samples with no false negative results and gave only 6.6 and 5.3% false positive rates, respectively. With meatmeal samples the ABS failed to detect one culturally positive specimen that was also positive by manual fluorescent antibody and gave one (1.1%) false-positive result. A high rate of false-negative results was obtained by ABS on unprocessed samples of creek water, poultry, and sausage. Adding another enrichment step to the protocol reduced the false-negative rate considerably but severely increased the false-positive rate. The instruments worked reasonably well, but research is needed to improve enrichment procedures for samples to be processed by the system.     

The Need for Routine Rubella Antibody Testing of Women

Results of rubella antibody tests performed by the California State Viral and Rickettsial Disease Laboratory on blood specimens collected in 1968 and 1969 from school children and women of childbearing age showed a slightly lower prevalence of rubella antibody in California than reported from most other areas of the United States. Among women of childbearing age, rubella hemagglutination-inhibition (hi) antibody was found in 72 percent of those tested in California compared with 80 percent to 90 percent in other areas of the country.Rubella antibody testing services offered by the State Virus Laboratory included situations in which a pregnant woman was exposed to a suspected case of rubella. It was shown that very few of these exposures constitute a significant risk to the fetus as most of the women already possessed antibody to rubella from past infection and in many instances the exposures were not to actual cases of rubella.The results of this study emphasized the urgency of obtaining blood specimens from pregnant women as soon as possible after exposure to rubella or development of symptoms of rubella. The urgency and anxiety attending these situations can largely be obviated if routine rubella antibody testing of women is carried out prior to pregnancy or at the first prenatal visit.     

Gradient Technique to Test the Effects of Substances on Fluorescent Antibody Reactions

A simple technique is described for making gradients of substances, applying them to bacterial cells during fluorescent antibody reactions, and observing their effects.     

Viable Cell Detection by the Combined Use of Fluorescent Glucose and Fluorescent Glycine

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.     

Detection of Salmonella by a Single-Culture Technique

Dulcitol-selenite enrichment medium in a motility flask was used for the detection of Salmonella in food. A drop in pH of the dulcitol-selenite enrichment motility broth indicated the presence of Salmonella; this phenomenon was confirmed by fluorescent-antibody staining. A complete correlation was found between fluorescent-antibody staining and recovery on Brilliant Green agar. Testing of 332 samples of 8 different kinds of foods and feeds indicated no significant difference in sensitivity between the new technique and a conventional Salmonella detection technique. The new technique permitted detection of even small numbers of Salmonella in 1 to 2 days.     

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.     

The Fluorescent Antibody Method in the Study of Immunopathologic Conditions

Histochemical studies of immunopathologic conditions were carried out, using Coons'' fluorescent antibody technique. Experimental conditions studied were: serum sickness, generalized anaphylaxis, the Arthus reaction and experimental glomerulonephritis. Human diseases studied were those referred to as “collagen diseases”. Specific immunologic reactants were localized in the lesions of all experimental conditions studied, thus offering objective evidence of a possible immunologic pathogenesis of the lesions. In human diseases, gamma globulin was localized in the lesions of rheumatic fever, rheumatoid arthritis, systemic lupus erythematosus and amyloidosis. Although the finding of gamma globulin in human lesions might suggest that it is an antibody, such an interpretation should be made with care since the gamma globulin could be deposited on a non-immunologic basis.The tissue-localizing properties of sera from different disease states showed appreciable variability within a given disease, as well as similar localizing properties among sera of different diseases. It is suggested that these serum factors (“autoantibodies”) might result as a host response and are not primarily involved in the pathogenesis of the disease.     

Comparison of Fluorescent Antibody with Cultural Technique for Isolation of Enteropathogenic Escherichia coli from Swine

In an investigation of hogs as possible reservoirs of human strains of enteropathogenic Escherichia coli (EEC), 92 six-month-old grain- and garbage-fed hogs were examined on the farm and again at the packing plant. Of the 331 specimens obtained by swabbing the rectum, cecum, and edible meat carcass of these hogs, 125 were presumptively positive for EEC when screened by the fluorescent-antibody (FA) technique. These “presumptive positive” specimens then underwent extensive bacteriological examination and complete serological typing. The FA technique proved to be an easier, simpler, and more economical procedure than culture when a large number of specimens were examined for possible EEC serogroups. It was found especially valuable for identification of multiple serogroups of EEC within a single specimen. It also appeared to be more sensitive than cultural examination, since results were not dependent on the presence of large numbers of organisms in the specimen, or even on their viability. However, the FA technique was found to be less specific than culture because of cross-reactivity with antigenically related Enterobacteriaceae when fluorescein-labeled antisera were used. Therefore, any specimen found positive on FA examination should be considered as presumptive positive until confirmed by bacteriological examination and complete serological study.     

A Comparison of the Fluorescent Antibody Method and a Standardized Cultural Method for the Detection of Salmonellas

The results of routine use of the indirect fluorescent antibody (FA) technique using the Spicer-Edwards H antisera set are reported for a range of agricultural and food samples. The FA technique was used on samples after the pre-enrichment incubation period in the proposed ISO method for isolation of salmonellas. The numbers of FA false positive samples ( ca. 5% overall) and FA false negative samples ( ca. 1·3%) were low, but some originally FA false positive results were later shown to be false negative cultural results.