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.     

Timed-Release Capsule Method for the Detection of Salmonellae in Foods and Feeds

A new method was developed for the detection of injured and uninjured salmonellae in foods and feeds. The steps of pre-enrichment in a nonselective broth and selective enrichment in a selective medium were combined into a single procedure. This was achieved by the gradual release of selective agents from wax-coated gelatin capsules added at the time of inoculation of nonselective basal broths. Pre-enrichment in lactose broth was combined with selective enrichment in tetrathionate or selenite-cystine broth by using timed-release capsules containing iodine or selenite. Five different categories of foods and feeds, naturally contaminated with salmonellae, were examined to compare the efficiencies of the capsule methods with conventional procedures. Combination of the separate steps of pre-enrichment and selective enrichment into a single procedure was feasible and resulted in substantial savings of labor and materials.     

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.     

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.     

Evaluation of a Fluorescent Antibody-Enrichment Serology Combination Procedure for the Detection of Salmonellae in Condiments, Food Products, Food By-Products, and Animal Feeds

The reliability of the enrichment serology (ES), fluorescent antibody (FA), and a combination of the FA and ES procedures for the detection of salmonellae were compared to the Salmonella cultural procedure outlined in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). A total of 126 subsamples from 22 different products were analyzed. By utilizing the BAM procedure as the reference standard, a total of 66 samples were positive for salmonellae. Within 44 h approximately 65% of the Salmonella-negative samples could be cleared by the FA test. At the end of 50 h 97% of the Salmonella-negative samples could be cleared by the combination FA-ES test. The FA procedure detected all 66 BAM positives but exhibited a high incidence of presumptive positives which were cultural negatives. The ES procedure detected 64 of the 66 BAM positives but exhibited a low incidence of presumptive positives which were cultural negatives. Incorporating positive FA and positive ES results in a combination FA-ES technique revealed that FA-ES positives were statistically equivalent to BAM positives.     

Evaluation of the Salmonellae Fluoro-Kit for Fluorescent-Antibody Staining

An evaluation of the newly developed Clinical Sciences, Inc. Salmonellae Fluoro-Kit, which attempts to standardize the various aspects of the fluorescent-antibody (FA) procedure, was performed with 120 naturally contaminated human food, animal feed, and raw material samples. The Association of Official Analytical Chemists (AOAC) method for the detection of salmonellae was used as the control method. The Fluoro-Kit was found to be simple and conveniento to use. The results of this preliminary study show an industrially acceptable rate of recovery of salmonellae by using the Fluoro-Kit in comparison with the A.O.A.C. method. The Fluoro-Kit shows promise as a rapid, salmonellae FA screening method. Problems originally encountered in the application of the Fluoro-Kit are discussed. According to the manufacturer, strict adherence to the now revised procedures included in the Fluoro-Kit will control these problems.     

Rapid Fluorescein and Protein Assay Method for Fluorescent-Antibody Conjugates

A method is presented for the rapid determination of the fluorescein content, protein content, and fluorescein-to-protein ratio for immune globulin conjugates with fluorescein isothiocyanate as the fluor. This method is based on the absorbance of the fluorescent antibody at those wavelengths primarily associated with the fluorescein and gamma-globulin fractions, and permits these materials to be determined by a single nondestructive analytical procedure. A small sample of the fluorescent antibody, in many cases 0.1 ml or less, is adequate for the above determinations. A nomograph is presented which allows simultaneous determination of the materials from the observed absorbance at the two wavelengths. The method is sufficiently accurate for most applications of the fluorescent-antibody techniques. Although this procedure has been developed primarily for fluorescent-antibody conjugates prepared from rabbit gamma-globulin, it can be used directly for antibodies prepared from other animals provided the gamma-globulin is relatively free from albumin.     

One-Day Fluorescent-Antibody Procedure for Detecting Salmonellae in Frozen and Dried Foods

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

A Rapid and Direct Plate Method for Enumerating Escherichia coli biotype I in Food

Direct enumeration of Escherichia coli biotype 1 in foods within 24 h has been achieved by a development of the method of Delaney, McCarthy & Grasso (1962) which is based on the production of indole at 44°. Indole positive E. coli growing at this temperature on a cellulose acetate membrane overlaying tryptone bile agar can be demonstrated. Characterization of 555 indole positive colonies from 843 samples of food showed that 95% were E. coli biotype 1 and a further 3·4% were 'faecal coliforms'. Anaerogenic and non-lactose fermenting E. coli are detected and the significance of these strains is discussed.     

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.     

Detection of Salmonellae in the Environment

The incidence of salmonellae in contrasting environments was compared in this study. Samples collected from or near surface waters in a lush hardwood forest yielded four salmonellae serotypes from six culturally positive samples. A total of 76 samples collected from the top of a granite outcropping over a 3-month period yielded 10 positive samples. Only two salmonellae serotypes were isolated, and one of these was isolated only once. The nature of the sample material had no significant effect on the detection of salmonellae from the two sampling sites. However, the presence or absence of visible moisture in the sample significantly affected the recovery of salmonellae. The results showed that even a harsh environment such as that found on top of Stone Mountain may serve as an ecological niche for the survival and transmission of salmonellae.     

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 Small Numbers of Airborne Bacteria by a Membrane Filter Fluorescent-Antibody Technique

Bacteria from stirred settling aerosols were recovered and identified by a rapid fluorescent-antibody technique previously described by Danielsson. Calibrated air samples from Serratia marcescens aerosols were drawn through a Millipore aerosol filter holder fitted out with a nonfluorescent membrane filter (type HAB 047). With the aid of a high-power incident-light ultraviolet microscope, the individual bacterial cells, trapped on the filter, could be inspected after staining with appropriate conjugate on the filter surface.     

Direct Fluorescent-Antibody Method for the Diagnosis of Pneumocystis carinii Pneumonitis from Sputa or Tracheal Aspirates from Humans

A direct fluorescent antibody (DFA) method was applied to sputum or tracheal aspirate from 68 patients with clinical or radiological evidence suggesting Pneumocystis carinii pneumonitis, and to 50 control patients. P. carinii was detected by DFA in specimens from 33 of the 69 clinical cases and 3 of the 50 controls. Specimens of lung from 11 of 33 DFA-positive cases were examined histologically, and 9 were positive. Four of 35 DFA-negative cases were examined histologically, and all were negative. Sputa or tracheal aspirates from 6 patients who were positive by both DFA and histological examination were examined also by methenamine silver staining; none could be diagnosed conclusively by this method. The results indicate that the DFA method is a sensitive and dependable procedure for the laboratory diagnosis of P. carinii pneumonitis in man.     

Indirect Fluorescent-Antibody Method for the Identification of Corynebacterium vaginale

The indirect fluorescent-antibody technique was employed in an attempt to develop a rapid method of identification of Corynebacterium vaginale. Six reference strains and ten clinical isolates selected on the basis of morphology and conventional biochemical tests were compared. Antisera were prepared in rabbits against the six reference strains. The most satisfactory antiserum was that prepared using strain 14018 grown diphasically (14018 Di) as the antigen. Certain of the antisera did exhibit a cross-reacting titer when reacted against Corynebacterium diptheriae, Corynebacterium xerosis, or Lactobacillus acidophilus. However, antisera adsorbed with these bacteria did not exhibit a significant decrease in titer when reacted against the homologous strain. Various other species of Corynebacterium as well as species of Nocardia, Actinomyces, Hemophilus, and Streptococcus did not fluoresce with the antisera. A specific antiserum was prepared by adsorbing anti-14018 Di with L. acidophilus. The adsorption removed the cross-reacting antibody but did not affect the staining reaction with C. vaginale strains. All reference strains and clinical isolates characterized as C. vaginale gave a definite positive reaction with the adsorbed anti-14018 Di. The specificity of the reactions was assessed by adsorbing the antiserum with the homologous strain. The data suggest that the indirect staining method will be of value in the rapid presumptive identification of C. vaginale.     

水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用

创伤弧菌是一种重要的食源性致病菌,主要存在于河口和海洋环境中,严重危害水产养殖业的发展和人类健康。建立快速、准确、易操作的检测方法对防控创伤弧菌的传染,保障水产养殖业发展和增强食品安全意义重大。基于创伤弧菌vvHA基因,利用一种新型的核酸扩增技术-环介导恒温扩增(loop-mediated isothermal amplification,LAMP),建立了创伤弧菌LAMP快速检测方法。对11种共46株细菌进行扩增,仅创伤弧菌为LAMP阳性结果,说明LAMP方法具有高度特异性。灵敏度试验结果表明,对创伤弧菌纯培养菌的检测灵敏度为15CFU/ml,对污染食品中创伤弧菌的检测灵敏度为24CFU/g。此法40~60min内即可完成检测,检验检疫实践证明:LAMP方法操作简便、特异性强、灵敏度高且成本低廉,具有良好的应用前景。     

Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 μm to remove large particles and of 0.22 μm to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 × 10² CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.     

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.     

一种可快速检测食品中酿脓链球菌和无乳链球菌的RPA方法的建立

为了实现食品中酿脓链球菌(Streptococcus pyogenes)和无乳链球菌(S.agalactiae)快速、高效检测,本研究建立了一种同时快速检测食品中这两种细菌的方法.本研究基于重组酶聚合酶等温扩增技术(recombinase polymerase amplification,RPA)原理,选择酿脓链球菌致...