Effect of formol on the fluorescence of staphylococci

Many strains ofStaphylococcus aureus give a strong immunofluorescence reaction with heterologous preparations of fluorescent antibodies. It is not known exactly whether this reaction is specific or unspecific. For diagnostic purposes it is important to reduce this fluorescence to low values without affecting the reaction of the homologous antigen with antibody. Formolisation of staphylococci, otherwise showing a strong fluorescence with the conjugates of heterologous antisera, for a longer period than 3 hours and at a formol concentration of 5%, lowers the fluorescence of the staphylococci to minimum. Formolisation proved suitable also when the staphylococci were contained in tissues (spleen, lymph nodes). In this case the fluorescence of the staphylococci disappears and the fluorescence of the tissues is suppressed. By treatment with formol in the concentrations used, the immunofluorescence reaction betweenPasteurella tuarensis and the corresponding preparation of the tularemy antiserum was not significantly suppressed.     

Use of the immunofluorescence method in an epidemic focus of tularaemia

Four hares found dead and 32 animals (small rodents, weasels, deer) caught in a focus of tularaemia were examined by the immunofluorescence method. In full agreement with the biological test, the use of fluorescent antibodies showed the presence ofPasteurella tularensis in the hares, even when the organs were massively contaminated with other bacteria, making cultivation tests quite impossible. In the other animals, all the test methods gave negative results. Tests of sera from 149 convalescent patients and 59 control subjects showed complete agreement (positivity or negativity) between the indirect immunofluorescence method and the agglutination reaction. The question of the titres found in the indirect immunofluorescence reaction are discussed. The first results with the use of fluorescent antibodies in practice thus confirm that this is a suitable method for examining animals in epizootic foci and for detecting specific antibodies in human serum.     

Fluorescent antibody demonstration ofPasteurella tularensis

The authors studied certain factors determining the possibility of the demonstration ofPasteurella tularensis by the immunofluorescence technique and the sensitivity, specificity and reliability of the reaction under experimental conditions. On the basis of the results they regard this method as suitable for rapid microbiological diagnosis (both for demonstratingPasteurella tularensis in material from patients and infected animals and for detecting antibodies in the serum of convalescents). The next step will be to study in greater detail the nature of the antigens and antibodies participating in the reaction and to verify the method in actual practice.     

Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS)

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.     

AMC-anti-FITC conjugates: novel reagents for amplified immunochemical techniques. Immunofluorescent staining of human fibroblasts

Summary Fluorescein antibodies were labelled with 7-aminocoumarin (AMC) derivatives, the 3-acetic acid and the 3-propionic acidN-hydroxysuccinimide esters. The labelled antibodies were used in conjunction with fluorescein isothiocyanate (FITC) and carboxyfluorescein-conjugated primary and secondary antibodies to develop novel immunofluorescent staining procedures. These methods combine the advantages of the fluorescence properties of AMC and the ready availability of FITC-labelled antisera to provide an amplified fluorescence signal as well as overcoming the photobleaching problems in FITC staining. The method is easy to perform and is expected to make an important contribution to the improvement of the quality of staining achieved with immunofluorescence. Details of the procedure used to stain human fibroblasts with antifibronectin antibodies are reported in order to illustrate the method.     

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 g ml^-1. Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30–70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - SBA soybean agglutinin     

Identification of H+/K(+)-ATPase alpha,beta-heterodimers.

Glutaraldehyde treatment of the C12E8 solubilized H+/K(+)-ATPase crosslinks the catalytic subunit with an apparent molecular mass of 94 kDa in SDS polyacrylamide gels into two Coomassie stained particles migrating at approx. 147 and 173 kDa. The subunit composition of these particles was determined from the comparative distribution of FITC fluorescence, wheat germ agglutinin and anti-beta antibody reactivity in control and crosslinked preparations. FITC exclusively labelled the catalytic monomer of the native preparation and its fluorescence was initially distributed into two broad bands centered at approx. 147 and 173 kDa after crosslinking. These fluorescent bands coincided with the Coomassie stained particles. A glycoprotein(s) detected by wheat germ agglutinin reactivity was present in diffuse areas between 65 and 86 kDa and 95 to 134 kDa in the control preparation. This area was also labelled by the anti-beta antibodies. With crosslinking, the distribution of the wheat germ agglutinin reactive protein and anti-beta antibodies coincided with the crosslinked particles labelled by FITC. The presence of both the catalytic monomer and the beta subunit glycoprotein in the crosslinked particles indicated that these proteins were closely associated in the C12E8 solution. This suggests that the minimal structural particle of the H+/K(+)-ATPase is an alpha,beta-heterodimer.     

On the Role of Disulfide Bonds in Polymerization of Soybean 7S Globulin during Storage

Modification of soybean 7S globulin with N-ethylmaleimide (NEM) was effective for preventing humidity-induced insolubilization during storage. The sulfhydryl-disulfide interchange reaction and/or oxidation of the sulfhydryl groups to form disulfide bonds closely related to the polymerization of the 7S globulin. A dimer, consisting of α′ and α subunits linked with disulfide bonds, was observed in the polymerized 7S globulin fractions, but this dimer was not readily apparent in the NEM-7S globulin. The formation of the dimer certainly initiates the insolubilization of the 7S globulin during storage. Although the β subunit had sulfhydryl groups, it did not take a part in the formation of the dimer.     

Antigens of Pasteurella tularensis: Preparative Procedures

Ether-water (EW) extraction of Pasteurella tularensis produced better antigens than five other chemical procedures. EW extracts produced from stationary-phase, liquid-grown, saline suspensions of strain SCHU S4 cells regularly induced agglutinin and precipitin formation in rabbits. Mice, guinea pigs, and monkeys also responded to EW extracts but with lower antibody levels. The use of strains of lower virulence, acetone-dried cells, organisms grown on a solid medium, and abbreviated extraction conditions all resulted in extracts with a diminished antigenicity, but logarithmic-phase and stationary-phase cells yielded equivalent EW extracts. The use of adjuvant, hyperimmunization, and large doses of antigen increased the precipitin responses of rabbits without appreciably altering the agglutinin response. By the appropriate combination of centrifugal fractionation of EW extracts, use of adjuvant, and vaccination schedule, rabbit antisera with either predominantly agglutinating or precipitating activities were obtained.     

Production of a tumor-specific xenoantiserum from partially purified immunoprotective tumor antigen

Summary Rabbits imunized with the immunoprotective TSTA fraction partially purified by preparative isoelectric focusing of 3 M KCl extracts from a chemically induced murine sarcoma, MCA-F, produced specific xenoantisera as assessed by an indirect membrane immunofluorescence assay. Only the immunizing tumor, MCA-F, and not the antigenically distinct MCA-D or MCA-T target cells were stained by the xenoantiserum. Absorption of anti-MCA-F antiserum with the antigenically distinct MCA-D or MCA-T cells did not reduce its capacity to bind to MCA-F cells. The immunofluorescence reaction was competitively inhibited by MCA-F fractions that induced specific immunoprotection: crude 3 M KCl extract, isoelectrically focused TSTA (fraction 15), and intact irradiated MCA-F cells. The TSTA specificity of these xenoantisera suggests that they may provide useful reagents for rapid isolation and characterization of the immunoprotective moiety. Abbreviations used: TSTA, tumor-specific transplantation antigens; MCA, 3-methylcholanthrene; MCA-F, MCA-D, MCA-T, methylcholanthrene-induced fibrosarcoma of C3H/HeJ mice; pIEF, preparative isoelectric focusing; PBS, 0.15 M phosphate-buffered saline (pH 7.2); MEM, minimum essential medium; MTD, minimum tumorigenic dose; Fr 15, fraction 15 of pIEF containing TSTA; FI, fluorescence index; FITC, fluorescein isothiocynate     

Virulence of representative Japanese Francisella tularensis and immunologic consequences of infection in mice

Francisella tularensis, which causes tularemia, is widely distributed in the Northern hemisphere. F. tularensis strains isolated in Japan are genetically unique from non‐Japanese strains; however, their phenotypic properties have not been well studied. Thus, mice were infected with representative Japanese strains of F. tularensis and their virulence and mouse immune responses to them assessed. Of four representative Japanese strains, the Ebina, Jap and Tsuchiya strains were susceptible to H₂O₂ and did not grow well intracellularly. Only Yama strain grew intracellularly and was lethal to mice. Infection with Yama strain resulted in drastic increases in IFN‐γ, CD4 and CD8 double‐positive T cells and Th1 cells (CD3, CD4 and Tim3‐positive cells), and a decrease in the ratio of CD8‐positive CD4‐negative T cells in mice. C57BL/6J mice that survived infection produced IgM antibodies to LPS and IgG2c antibodies to 43, 19 and 17 kDa proteinase K‐sensitive components. These data are valuable for understanding the phenotypic properties of F. tularensis in Japan.     

Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa gamma G and gamma M-antibodies during prolonged immunization

Pike, Robert M. (University of Texas Southwestern Medical School, Dallas), Mary L. Schulze, and Cleo H. Chandler. Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa gammaG and gammaM antibodies during prolonged immunization. J. Bacteriol. 92:880-886. 1966.-Antibody produced in rabbits immunized with acetone-dried typhoid bacilli was followed over a period of 445 days by agglutination and by quantitative precipitation. Repeated injections of vaccine resulted in suppression of antibody titers. Both gammaG and gammaM antibodies were rapidly increased by booster injections after rest periods during which titers had decreased to low levels. The O agglutinin titers and the amount of antibody protein, as determined by precipitation with endotoxin, generally were parallel, except in serum specimens in which unusually large proportions of the agglutinating activity were found in the gammaG fraction. These exceptions were explained by the greater agglutinating capacity of the gammaM. Endotoxin precipitated about 10 times as much antibody from gammaG preparations as it did from gammaM fractions of equivalent agglutinating strength. A much higher proportion of the serological activity, therefore, was found in the gammaG fractions when antibody was measured by precipitation than when agglutination was used as the measure of activity.     

Wall structure of the Neurospora hyphal apex: immunofluorescent localization of wall surface antigens.

Antisera have been raised in rabbits against three wall fractions from Neurospora crassa. Fractions were separated according to Mahadevan & Tatum (1965), i.e. fraction I, glucan-peptide-galactosamine complex; fraction III, laminarin-like glucan; and fraction IV, chitin. Distinct patterns of immunofluorescent staining were obtained using an indirect staining method. Hyphae stained with antiserum to fraction I showed maximum fluorescence in the apical and/or subapical regions: in both cases, fluorescence showed a sharp decrease with distance behing the subapical region. Hyphae stained with antiserum to fraction III showed faintly fluorescent tips with fluorescence increasing with distance from the tip. Hyphae stained with antiserum to fraction IV showed faint fluorescence, equivalent to levels of autofluorescence, except at the sites of hyphal fractures. Antisera were also raised against whole walls from 24 and 120 h cultures. Hyphae stained with antisera against whole walls which had previously been absorbed to remove antibodies to fractions I, III, and IV showed preferential staining of apices. The uncharacterized tip antigen(s) thus revealed was also demonstrated on immunodiffusion plates. This pattern of immunofluorescence was compared to the fluorescence of apices after staining with an optical brightener. Enzymic dissection procedures did not generally give reliable results with apices from 24 h cultures. Untreated apices appeared amorphous, while a drastic chemical treatment revealed randomly oriented microfibrils which were shown to be alpha-chitin. The apical hyphal walls were significantly thinner than those from more mature hyphal regions.     

Failure to Detect Cell-Associated Enterotoxin B in Staphylococcus aureus by Immunofluorescence

Enterotoxin B-producing and -nonproducing Staphylococcus aureus strains showed cell fluorescence when tested with fluoresceinisothiocyanate-labeled rabbit anti-enterotoxin B globulin, probably as a result of a protein A-immunoglobulin G (Ig G) interaction. No cell-bound enterotoxin B could be detected by immunofluorescence using F(ab(1))(2)-fragments of anti-enterotoxin B globulin. However, soluble enterotoxin B could be estimated by immunofluorescence. Approximately 1,000-fold more enterotoxin B was detected by immunodiffusion as an extracellular product in the media than could be detected in the cell fraction. The results show that intact Ig G is not suitable for the detection of antigens other than protein A on the cell surface of S. aureus in conventional immunofluorescence. For such purposes, the use of F(ab(1))(2)-fragments of Ig G is recommended.     

Humoral antibody response and Ig characterization of the specific agglutinins in rabbits during experimental American trypanosomiasis

A comparative follow up study of the specific agglutinins detected by direct agglutination (DA) test and the immune response detected by specific lysis (SL), indirect immunofluorescence (IFA), indirect hemagglutination (IHA) and complement fixation (CF) tests in rabbits inoculated with trypomastigotes of T. cruzi is reported here.The specific antibody response was detected first by DA test. Reductive cleavage of sera with 2-mercaptoethanol produced a drop in the agglutinin titer of the sera during the first 30 days of infection.The next test to become positive was SL and later on the IFA, IHA and CF tests became positive simultaneously.When fractions obtained by column chromatography in Sephadex G-200 were tested serologically it was demonstrated that specific antibodies were detected mainly in fraction I (IgM) of the pooled rabbit sera obtained 15 days after inoculation (acute stage), and in fraction II (IgG) of the pooled sera obtained from rabbits 90 days after inoculation (chronic stage).Antigens prepared with trypsinized and formolized epimastigotes of three T. cruzi strains, belonging to each one of the different immunological groups described, worked similarly in the detection of specific agglutinin antibodies.Trypanosoma cruzi agglutinins were highly specific in their reaction with their homologous T. cruzi antigens as was proved by the low agglutinin titer obtained in sera from infected rabbits when, instead of T. cruzi epimastigotes, promastigotes of L. donovani were used as antigen, and by the incapacity of this parasite to absorb the T. cruzi agglutinins.     

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.     

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.     

Direct immunofluorescence of plant microtubules based on semiconductor nanocrystals

Fluorescence microscopy in combination with multiple, simultaneous labeling of biomolecules has been a key breakthrough in cell biology. However, the spatiotemporal resolution of this approach is limited by bleaching of the fluorescence label and illegitimate cross-reference of the label. CdSe-based semiconductor nanocrystals with their excellent bleaching stability would be an alternative to overcome this limitation. We therefore explored direct immunofluorescence based on nanocrystal-conjugated antibodies using plant microtubules as model. We compared two strategies of bioconjugation, covalent coupling of antitubulin antibodies to BSA-coated nanocrystals and covalent coupling to nanocrystals that were surrounded by functionalized silica shells. Both nanoparticle-antibody conjugates were used to follow the dynamic reorganization of microtubules through the cell cycle of a tobacco cell culture in double and triple staining with FITC as conventional fluorochrome and Hoechst 33258 as marker for mitotic duplication of DNA. BSA-coated nanocrystals visualized fluorescent dots that decorated the various arrays of microtubules. The specificity of the antibody was maintained after conjugation with the nanocrystals, and the antibodies correctly represented the dynamics of cell-cycle-dependent microtubular reorganization. However, this approach did not yield a contiguous signal. In contrast, silica-shelled nanocrystals visualized contiguous microtubules in the same pattern as found for the conventional fluorochrome FITC and thus can be used as labels for direct immunofluorescence in plant cells.     

Lipopolysaccharides in four strains of the unicellular cyanobacterium Synechocystis

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir⁺) which react with FA from Sp7 the type strain. None of the nir^- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir^- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.Abbreviations Fa fluorescent antibody - FITC fluorescein isothiocyanate - Rh ITC gelatin-rhodamine isothiocyanate - nir⁺ nitrite reductase positive - nir^- nitrite reductase negative     

Characterization of zoospore and cyst surface structure in saprophytic and fish pathogenicSaprolegnia species (oomycete fungal protists)

Summary The importance of the surface structure and chemistry in zoospores and cysts of oomycetes is briefly reviewed and the organelle systems associated with encystment described. The surface structure and chemistry of primary and secondary zoospores and cysts ofSaprolegnia diclina (a representative saprophytic species) andS. parasitica (a representative salmonid fish pathogen) were explored using the lectins concanavilin A (Con A) and wheat germ agglutinin (WGA) and monoclonal antibodies (MAbs) raised against a mixed zoospore and cyst suspension ofS. parasitica. The binding of lectins and antibodies to spores was determined using immunofluorescence microscopy with fluorescein isothiocyanate-labelled probes and with electron microscopy with gold-conjugated probes applied to spore suspensions post-fixation. In both species Con A, which is specific for glucose and mannose sugars, bound to both the surface of primary and secondary zoospores (the surface glycocalyx) and their cyst coats and readily induced zoospore encystment. The binding to the cysts appeared to be mainly associated with the matrix material released from the primary and secondary encystment vesicles and which appeared to diminish with time. No binding to germ tube walls was observed with this lectin. The MAb labelling showed a generally similar binding pattern to the primary and secondary cysts to that observed with Con A, although the binding to zoospores was more variable. Primary zoospores bound the antibodies but secondary zoospores appeared less reactive. It is suggested that the MAbs share a common epitope with one or more of the Con A-binding components. In both species WGA, which is specific for amongst other things the sugar N-acetyl glucosamine, bound to localised apical patches on the primary zoospores. This lectin also binds to the ventral groove region of secondary zoospores ofS. diclina, which were induced to encyst by this lectin. In contrast secondary zoospores ofS. parasitica were not induced to encyst by the addition of WGA and showed a patchy dorsal binding with this lectin. WGA also binds to both the inner wall of discharged primary cysts and the young germ tube walls of both species. These observations are discussed both in relation to other oomycete spores and to their possible functional and ecological significance.Abbreviations BSA bovine serum albumin - Con A Concanavalin A - DBA Dolichos biflorus agglutinin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - EV encystment vesicles - FCS foetal calf serum - FITC Fluorescein isothiocyanate - FV peripheral fibrillar vesicles - G+F 0.2% glutaraldehyde and 2.0% formaldehyde primary fixative solution - 2G 2% glutaraldehyde primary fixative - LM light microscopy - MAbs monoclonal antibodies - LPV large peripheral vesicles - PBS phosphate buffered saline - PCV flattened peripheral cisternae - PEV primary encystment vesicle - PIPES piperazine-N,N1-bis(2-ethane sulfonic acid) - PNA Ricinus communis agglutinin - RAM-FITC/Au_10–20 Fluorescein isothiocyanate/gold (10 or 20 nm) labelled rabbit anti-mouse immunoglobulin - RCA Ricinus communis agglutinin - SEM scanning electron micrograph - SBA soybean agglutinin - SEV secondary encystment vesicles - TEM transmission electron micrograph - UEA I Ulex europaeus agglutinin - WGA wheat germ agglutinin