Bacterial adhesiveness and invasiveness in cell culture monolayer.
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Adhesiveness↗
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Location of U.V.-absorbing substance in isolated skeletal muscle fibres. The effect of stimulation.
1. In an attempt to study the intracellular location of ATP in skeletal muscle the distribution of substances which absorb light at 260 nm wave-length has been studied in isolated muscle fibres with the aid of a modified U.V.-microscope.2. U.V.-absorption in resting frog fibres was found to be higher in the I band than in the A band which confirms earlier findings. In stretched fibres (sarcomere length 2.9-3.6 mum) an absorbing substance could be seen to be concentrated in a pair of narrow lines, centred at the Z-line. The separation of the lines increased with increasing sarcomere length.3. Snake fibres, with sparse triads located at the A-I junction, displayed an absorption pattern very similar to that of frog fibres. It is concluded that it is unlikely that the absorbing substance is associated with the sarcotubular system.4. The absorption pattern of frog fibres remained unchanged during a tetanus. No clear changes could be detected after a period of stimulation, neither after single twitches nor after repeated tetani.5. In further attempts to cause exhaustion, metabolically poisoned fibres were stimulated repetitively until they went into rigor. The absorption pattern was essentially unchanged also when rigor tension started to develop.6. The characteristic absorption pattern was observed also in glycerol-extracted fibres. It was confirmed by spectrophotometry that glycerol-extraction led to the disappearance of a large amount of a substance with the spectral characteristics of ATP.7. The higher U.V.-absorption in the I band does not prove that the major part of the ATP in the fibre is concentrated here; the absorption could either be due to a minor fraction of the ATP or to RNA.
Absorption↗
Microdamage accumulation in bovine trabecular bone in uniaxial compression.
In this study we investigated how microdamage accumulated with increasing compressive strain in bovine trabecular bone. We found that little damage is created in the linear elastic region, up to -0.4 percent strain. At an average strain of -0.76 percent +/-0.25 percent, the stress-strain curve became nonlinear, and peaked at -1.91 percent +/-0.55 percent strain. Microdamage increases rapidly during the peak of the stress-strain curve, and a localized band of damage formed. At strains beyond the ultimate strain, the damaged band widened and the density of damage within the band increased. Microdamage occurred as groupings of cracks; the majority of damage occurred as regions of cross-hatching. All microdamage parameters increased with increasing maximum compressive strain. We also observed exponential relationships between crack numerical density and damage (1(o) - (o)Esec/E0) and between crack length density and damage.
Animals↗
Quantification of the horny layer using tape stripping and microscopic techniques.
Tape stripping is a method well suited for studying the penetration of topically applied substances into the upper part of the skin, the stratum corneum (SC). The amount of topically applied substances removed by each tape strip can be determined by common analytical methods. These amounts have to be correlated with their location within the horny layer in order to determine penetration profiles. Therefore the amount of SC on each removed strip must be determined. In the study reported, this amount was derived from the covering density of the SC cells, the corneocytes, on tape strips using classic microscopic techniques and laser scanning microscopy. The amount of corneocytes determined by both microscopic methods shows a good correlation (R2 +/- S.D.= 0.95 +/- 0.02) with the pseudoabsorption of the corneocytes, a method usually used to determine the SC amount on the removed strips. These measurements require the application of a special spectrometer, whereas the covering density can easily be determined using widely available microscopic equipment. The penetration of a typical UV filter, butyl methoxydibenzoylmethane, applied at two different formulations, was studied using the microscopic method developed. The calculated penetration profiles show that the applied formulation influences the amount of corneocytes on the removed strips.
Adult↗
Terminal transferase-dependent PCR (TDPCR) for in vivo UV photofootprinting of vertebrate cells.
Terminal transferase-dependent PCR (TDPCR) is a versatile, sensitive method for detecting DNA lesions such as those generated by the footprinting agents commonly used to detect in vivo protein-DNA interactions. Data similar to those obtained by ligation-mediated PCR (LMPCR) are obtained, but one advantage of TDPCR is that no special enzymes are needed other than terminal deoxynucleotide transferase, T4 DNA ligase, and thermostable DNA polymerases. A detailed TDPCR protocol is given for using UV photofootprinting to detect in vivo footprints and chromatin fine structure in vertebrate cells. One version of the protocol makes use of nonradioactive labeling by near-infrared fluorochromes and detection by a LI-COR DNA sequencing instrument. Sensitivity similar to that of (32)P-labeling is obtained, but with superior band resolution and quantitation.
Animals↗
Affinity of cellular constituents of two bacteria for fluorescent brighteners.
Two fluorescent brighteners were used to stain an isolate of Bacillus cereus var. mycoides and soil pseudomonad. The stained organisms were fractionated by two procedures to determine which cellular constituents were reacting with the brighteners. Both fractionation procedures provided evidence that the brighteners were adsorbed to proteins within the cells. Microscopy examination of ghost cells of the bacillus showed that cell walls were not being stained. Spheroplasts of the bacillus and the pseudomonad were stained by the brighteners.
Bacillus cereus↗
Examination of isolated yeast cell vacuoles for active transport.
Isolated vacuoles of the yeast Candida utilis did not show active transport of S-adenosylmethionine, uric acid, and several amino acids which they concentrate in vivo.
Arginine↗
Agglutination of human erythrocytes by Fusobacterium nucleatum: factors influencing hemagglutination and some characteristics of the agglutinin.
Various aspects of agglutination of human erythrocytes by fusobacterium nucleatum were examined. Titration experiments done in buffered saline at pH 6 to 10 showed the same agglutination endpoint. The presence of high concentrations of NaCl in reaction mixtures did not alter titers, but KCl in concentrations of 0.5 to 3.6 M increased titers twofold. The agglutinin was inactivated by heat, acid, alkali, 5% Formalin, and the proteolytic enzyme subtilopeptidase A and therefore appeared to be a protein. Treatment of bacterial cells with 2-mercaptoethanol had no effect. Hemagglutination inhibition experiments revealed that arginine and other compounds containing a guanido group as part of their structure were inhibitory at low concentrations. Various hexoses, some hexose derivatives, and most common metal ions, when added to bacterium-erythrocyte mixtures, had no effect. The binding of dansyl-L-arginine to bacteria, but not erythrocytes, was demonstrable by ultraviolet fluorescence microscopy.
Agglutinins↗
Calcofluor staining of cellulose during microcyst differentiation in wild-type and mutant strains of Polysphondylium pallidum.
Calcofluor White ST was used to monitor the morphological events in the biogenesis of cellulose in the microcyst wall of the wild-type strain (WS-320) and two developmental mutants (mic-1 and mic-2) of Polysphondylium pallidum. During encystment, the cell surface acquires a Calcofluor-specific material which appears to be cellulose because of its sensitivity to purified cellulase. Cellulose-containing vesicles appear distributed throughout the cytoplasm of encysting cells of the three strains. Later, the cellulose-rich vesicles appear near the cell surface. Subsequently, the cell surface stains with Calcofluor, and the vesicles are no longer detectable. Intracellular vesicles resembling the cellulose-rich vesicles in size, in the timing of appearance, and in cellular location are also seen in thin sections. These vesicles are surrounded by a single unit membrane, and their amorphous matrix, which contains a dense irregular core, further implicates them as the basis for the bilayered microcyst wall.
Benzenesulfonates↗
Large bodies of Mycoplasma and L-form organisms.
The large bodies of various Mycoplasma and L-form organisms were studied by ultraviolet fluorescence microscopy of preparations stained with various fluorochromes. Primuline and Thioflavine S specifically stained the outer portion or rim of the large bodies, and the fluorescence characteristics of the stained bodies differed from those for other microorganisms and for spheroplasts and protoplasts. Small granular structures similar in size and morphology to minimal reproductive units were observed within some of the large bodies by phase microscopy and by fluorescence microscopy with acridine orange or Coriphosphine O. Micromanipulation probing of the large bodies revealed their elastic nature; many of the large bodies could be subdivided into two or more smaller circular bodies, each retaining the fluorescence staining properties of the parent body. Under these conditions, however, a few of the large bodies were ruptured, leaving the stainable outer boundary area as a stable residual structure. The large bodies were somewhat resistant to various rigorous treatments normally employed to eliminate viability of Mycoplasma and L-form cultures. Structures similr to large bodies were observed in various natural tissues, and structures resembling large bodies in size, morphology, fluorescence staining characteristics, and reaction to micromanipulation probing were reconstructed from an acetone extract of egg yolk. Overall, the large bodies of Mycoplasma and L-form organisms appeared to be structures resulting from accumulations of metabolic by-products and medium components within or on which minimal reproductive units had become entrapped, although it could not be ruled out that they might be defined structures specifically formed during culture as protective lipoidal sacs for the minimal reproductive units.
Bacteria↗
Use of UV ParaLens adapter for detection of acid-fast organisms.
Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears.
Benzophenoneidum↗
A light and electron microscopic study of the liver in case of erythrohepatic protoporphyria and in griseofulvin-induced porphyria in mice.
The light- and electron-microscopic appearances of a liver biopsy from a case of erythrohepatic protoporphyria are compared with the appearances of the liver in griseofulvin-induced porphyria in mice. Crystals are present in hepatocytes and in the bile secretory pathways in each case. The nature and significance of these is discussed.
Adult↗
Early detection of Chlamydia trachomatis using fluorescent, DNA binding dyes.
HeLa 229 cells were infected with genital tract strains of Chlamydia trachomatis. After incubation for varying times the infected cells were fixed and stained with the fluorescent DNA binding dyes Hoechst 33258 or DAPI for comparison with conventional Giemsa stain. Fluorochrome-treated preparations were examined by incident ultraviolet fluorescence microscopy and the Giemsa-stained preparations by dark-ground light microscopy. Chlamydial inclusion bodies could be identified unambiguously as early as 18 hours after infection of HeLa 229 cells using either Hoechst 33258 or DAPI but not until some 48 hours in Giemsa-stained preparations. The DNA rich chlamydial elementary bodies in infected egg yolk suspension were readily detected using Hoechst 33258. The fluorescent dye technique was simpler and more rapid than Giemsa staining. Using Hoechst 33258 it is possible to speed up the identification of chlamydial isolates growing in tissue culture.
Benzimidazoles↗
Diagnosing Pneumocystis carinii pneumonia by cytological examination of bronchoalveolar lavage fluid: report of 15 cases.
Sixty episodes of pneumonia occurring in 53 immunosuppressed patients were investigated by bronchoalveolar lavage. Pneumocystis carinii was diagnosed on 15 (25%) occasions. In all cases the Papanicolaou stained lavage fluid presented a distinctive appearance and contained abundant, often biphasic, staining, "honeycomb" debris, and few alveolar macrophages. The Grocott methenamine silver technique confirmed the presence of characteristic cystic organisms in the debris in all 15 instances. Cysts containing internal sporozoites were identified in Gram stained material only with difficulty. Neither May-Grünwald-Giemsa stain nor fluorescence microscopy under ultraviolet light were effective for routine investigation.
Adolescent↗
Class I and class II major histocompatibility complex antigens on hepatocytes: importance of the method of detection and expression in histologically normal and diseased livers.
Methodological differences in major histocompatibility complex (MHC) antigen detection were investigated on isolated, viable hepatocytes and cryostat hepatic sections from 27 children with liver disorders, six of whom had normal histology. Class I antigens were constantly found on sections using a three step immunoperoxidase technique after acetone/chloroform fixation, other techniques being less sensitive, or on isolated hepatocytes by indirect immunofluorescence alone. With mechanical isolation the percentage of positivity ranged from 85 to 100%, while with collagenase isolation it ranged from 22 to 49% on immediate testing, and from 53 to 80% after 24 hour incubation. Class II antigens were only detected in one patient with autoimmune chronic active hepatitis and two with primary sclerosing cholangitis. Flow cytofluorimetric analysis in 11 cases confirmed class II or class I positivity, or both, on isolated hepatocytes, allowing MHC antigen expression on hepatocytes to be measured. Class I and II antigen detection on hepatocytes is influenced by the technique used. Although class I antigens are invariably expressed on hepatocytes, class II antigens are only found on hepatocytes from patients with immune mediated liver disorders.
Adolescent↗
Evaluation of an enzyme immunoassay kit for detecting cryptosporidium in faeces and environmental samples.
AIMS: To evaluate a commercially available enzyme immunoassay based on a monoclonal antibody to a genus specific Cryptosporidium (IDEIA Cryptosporidium; Dako) antigen for detecting Cryptosporidium oocysts in faecal and environmental samples. METHODS: 435 human faecal samples and post-filtration deposits from 10 reservoir samples, and from six tap water samples seeded with Cryptosporidium oocysts, were examined by EIA according to the manufacturer's instructions, and by microscopic examination of phenolauramine stained smears. Samples giving discrepant results were examined by specific immunofluorescence, before and after concentration of oocysts. RESULTS: Sixteen (3.6%) faecal samples were positive by both microscopy and EIA; five (1.1%) were positive by microscopy of auramine-phenol stained smears (but were not confirmed by specific immunofluorescence) and negative by EIA; one (0.2%) was positive by EIA alone, but confirmed by specific immunofluorescence; and 362 (83.2%) were negative by both microscopy and EIA. Compared with immunofluorescence positive faecal samples, the sensitivity of conventional microscopy and EIA were 94% and 100%, and specificity 76.4% and 100%, respectively. Fifty one (11.7%) were not examined by microscopy due to detection of other pathogens in a previous sample from that patient, but were found to be negative by EIA. Ten reservoir water samples (not suspected of being linked to cases of cryptosporidiosis) were negative by both microscopy and EIA. Of six samples of tap water seeded with varying concentrations of Cryptosporidium oocysts, two (10(2) and 10(3) oocysts/l) were positive by both microscopy and EIA, two (10 and 1/l) by EIA alone, and two (0.1/l and unseeded water) were negative by both microscopy and EIA. CONCLUSIONS: The kit is simple and rapid to use and offers a less subjective method than microscopy for detecting Cryptosporidium in faecal samples submitted to a busy diagnostic laboratory.
Animals↗
A satisfactory medium and technique for the mass cultivation of Sphaerophorus necrophorus.
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Anaerobiosis↗
Rapid diagnosis of catheter-related sepsis using the acridine orange leukocyte cytospin test and an endoluminal brush.
BACKGROUND: In neonates, the acridine orange leukocyte cytospin (AOLC) test has been found to be a highly sensitive test for the detection of infected i.v. catheters in situ, which provides a result in less than 1 hour. Preliminary data suggested that the AOLC test was of limited value in adults. We report here a modification of the test for adult patients with indwelling central venous catheters. METHODS: A prospective study was performed on two groups of 50 adult patients with suspected sepsis and a central venous catheter. The AOLC test was carried out after the clinical decision to remove the catheter had been made. In group 1 patients, a blood sample was withdrawn from the catheter for the AOLC test. In the patients in group 2, an endoluminal brush was used to "sweep" the catheter before the collection of the blood sample. Results of the AOLC test were compared with culture of the removed catheter tip. RESULTS: From the catheters in group 1 (no brush), 17 catheter tips were found to be infected, but the AOLC was positive in only two patients (12%). In group 2 (brush), 18 tips were infected, and the AOLC test was positive in 15 patients (83%). The use of the endoluminal brush significantly improved the yield of the AOLC test (p < .01) to levels reported in neonates. The AOLC test produced no false positives in either group CONCLUSION: When used independently, the AOLC test was not sensitive enough to detect catheter-related sepsis. However, in combination with an endoluminal brush, the AOLC test was much more sensitive and has the potential to provide a simple, rapid, and accurate diagnostic test for catheter-related sepsis, which does not require removal of the catheter.
Acridine Orange↗