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.     

Rapid Fluorescent-Antibody Stain Technique with Group A Streptococci

A rapid fluorescent-antibody stain technique used with young broth cultures of trypsinized group A streptococci is described. Results from 1,214 trypsinized cultures of group A streptococci employing the rapid stain method were equivalent, both in specificity and sensitivity, when compared to results obtained from identical non-trypsinized cultures stained with the longer standard technique of Cherry, Goldman, and Carski. Moreover, the rapid method requires less than 2 min to complete.     

Soluble Antigen Fluorescent-Antibody Technique

An indirect fluorescent-antibody (FA) procedure employing soluble antigen fixed onto an artificial matrix was developed, and a mechanical means for reading of test results was devised. The method employs two small cellulose acetate paper discs for each test. One disc contains soluble antigen diluted in 1% bovine serum albumin (BSA); the other contains only 1% BSA and serves as a control. After testing by the indirect FA procedure, the results of the tests are read on a fluorometer fitted with a paper chromatogram door. The instrument is set at zero with the control disc as a blank, and the specific fluorescence of the antigen disc is determined. Findings obtained with homologous and heterologous antisera indicated that the method yields excellent results. The soluble antigen fluorescent-antibody technique has definite advantages over the conventional indirect FA procedures. (i) The investigator may objectively select the antigen to be employed. (ii) It is possible to obtain objective mechanical reading of test results rather than the highly subjective readings required by conventional methods. (iii) The system compensates for any nonspecific fluorescence contributed either by the serum (e.g., drugs) or by free fluorescein in the conjugated antiserum.     

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 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.     

A Rapid Safranin-Metal Phthalocyanine Double Staining Technique for Plants

Pure metal 4.4',4',4'-tetxa-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4',4'-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4',4'-tetra-sulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log ε = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.     

Simplified Fluorescent-Antibody Staining Method for Primary Plate Isolates of Group A Streptococci

Beta-hemolytic streptococci were identified from primary plates by using a simplified fluorescent-antibody staining method. There was 100% correlation between this method and standard precipitin grouping.     

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.     

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.     

A Rapid Silver Staining and Destaining Technique for the Nucleolus Organizer Region

Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.     

A Simple and Rapid Staining Technique for Plastic Embedded Cartilage and Bone

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semitbin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na₂ CO₃, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.     

Study of Developmental Stages of Methylosinus trichosporium with the Aid of Fluorescent-Antibody Staining Techniques

When stained by using an indirect fluorescent-antibody technique, Methylosinus trichosporium displayed an uneven fluorescence. Exospores and the polar tips of some vegetative cells displayed a more intense fluorescence than the other cells. Cross-absorption of the specific anti-M. trichosporium immunoglobulin G with exospores resulted in no fluorescence of exospores or exospore regions of sporulating vegetative cells. This demonstrated that antigens were present on exospores and exospore regions of vegetative cells that are different from vegetative cell antigens. Taking advantage of this phenomenon, three fluorescentantibody staining techniques were developed which were used to study the life cycle of M. trichosporium.     

An Indirect Radiolabelled Antibody Staining Technique for the Rapid Detection and Identification of Bacteria

Small numbers of Bacillus subtilis var. niger spores, Serratia marcescens and Francisella tularensis can be detected and identified in an hour using an indirect radiolabelled antibody staining technique with homologous rabbit antibacterial antibody and radiolabelled goat anti-rabbit globulin. Tritium is better than ¹²⁵I, ¹⁴C and ³⁵S for labelling the globulin. The reagent obtained is sensitive and adequately stable.     

Evaluation of the Indirect Fluorescent-Antibody Technique for Identification of Naegleria Species

The indirect fluorescent-antibody technique was used to assess a rapid method for identification of amoebae belonging to the genus Naegleria. Thirty-eight Naegleria and eight other limax amoeba strains were examined by using one N. gruberi and two N. fowleri antisera. All pathogenic Naegleriae, most of which originated from fatal cases of primary amoebic meningo-encephalitis, were identified as belonging to the fowleri species. Most of the N. gruberi strains showed irregular fluorescence. Other limax amoebae, such as Vahlkampfia, Acanthamoeba, Hartmannella, and Schizopyrenus sp. gave negative responses with the prepared antisera. The indirect fluorescent-antibody technique allows the identification of N. fowleri in a mixed culture of both N. fowleri and N. gruberi strains. Twenty-two Naegleria isolated from a suspected stream, other surface waters, and muddy soil could be excluded from the fowleri species with the indirect fluorescent-antibody technique. The results obtained demonstrate that this immunological technique is a valid method for the rapid identification of N. fowleri trophozoites.     

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.     

A New Staining Technique For Microfilariae

A rapid whole mount staining method is described to identify and differentiate microfilariae without elaborate processing. A single solution combining Hoyer's mounting medium and hematoxylin stain facilitates light microscopic examination of nuclei and sheaths of microfilariae. The new technique stains microfilariae adequately in three to seven minutes at 60-64 C making the method preferable to conventional methods that may take as long as 45 to 60 minutes. Lantern heat may be used to heat slides in rural areas with good results.     

Rapid Nuclear Staining Method for Saccharomyces cerevisiae

Mithramycin was used to stain nuclei in mitotically dividing and sporulating yeast.