A simple fluorescence of staining technique for in situ soil microorganisms.

A simple, rapid straining technique using the magnesium salt of 1-anilino-8-naphthalene sulfonic acid is described. Treatment of soil with an aqueous, membrane-filtered solution (3.5 mg/ml) of the salt causes the soil microorganisms to fluoresce when examined with light from a mercury arc light source.     

Metabolic effects of some electrofluorimetric dyes

The effect of five electrofluorimetric dyes on mitochondrial metabolism was examined to determine their suitability for mitochondrial studies and other biological uses. The dyes merocyanine 540, 8-anilino-1-naphthalene sulfonic acid and bis(1,3-dibutyl barbituric acid-(5))-pentamethane oxonol were found to be inhibitors of the respiratory chain. However, the first two exerted their effect only at high concentrations. 3.3′-Dihexyl-2,2′-oxacarbocyanine was found to act as an uncoupler. 3,3′-Dipropyl-thiocarbocyanine inhibited β-hydroxybutyrate respiration while dissociating succinate supported respiration from the phosphorylation of ADP. Merocyanine 540 and 8-anilino-1-naphthalene sulfonic acid may be the best suited for studies of membrane potentials in mitochondria since their effect on metabolism is negligible.     

A method for the detection and analysis of growth patterns of microorganisms in soil.

A fluorescence-staining technique using the magnesium salt of 8-anilino-1-naphthalene sulfonic acid is described and used to follow the changes in the distribution patterns of microorganisms in soils. A statistical procedure was used to determine the occurrence of significant differences in clumping of bacteria (i.e., production of colonies) in different regions of artificial soil-aggregate systems treated with nutrient solutions and also with a herbicide, Linuron. The response of soil microorganisms to glucose amendment was most marked in the aerobic, outer zone of aggregates. Linuron inhibited colony formation in aggregates treated with the herbicide. The method allows continued observations to be made on the same soil sample at intervals during incubation and os can be used to determine growth rates, inhibitory effects of chemicals, distribution patterns in soils, effects of added nutrients, and other effects where growth in situ is important.     

A direct observation technique for studies on rhizoplane and rhizosphere colonization

Summary A slide incubation chamber was described which allowed small plants to be grown from seed and the root systems to be observed microscopically. A fluorescence stain, the ammonium salt of 8-anilino-1-naphthalene sulfonic acid, was applied to the soil in which the roots were growing and the stained microorganisms on the roots and in the rhizosphere were counted. A statistical pattern analysis technique, the two-within-four randomization test, was used to analyze the data obtained from quadrats on the roots. Distinct colonization patterns and colony growth, especially of bacteria, were easily distinguished with the technique.     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monensin-based medium for determination of total gram-negative bacteria and Escherichia coli.

Plate count-monensin-KCl (PMK) agar, for enumeration of both gram-negative bacteria and Escherichia coli, is composed of (per liter) 23.5 g of plate count agar, 35 mg of monensin, 7.5 g of KCl, and 75 mg of 4-methylumbelliferyl-beta-D-glucuronide (MUG). Monensin was added after the medium was sterilized. The diluent of choice for use with PMK agar was 0.1% peptone (pH 6.8); other diluents were unsatisfactory. Gram-negative bacteria (selected for by the ionophore monensin) can be used to judge the general quality or sanitary history of a commodity. E. coli (differentiated by its ability to hydrolyze the fluorogenic compound MUG) can be used to assess the safety of a commodity in regard to the possible presence of enteric pathogens. Pure-culture studies demonstrated that monensin completely inhibited gram-positive bacteria and had little or no effect on gram-negative bacteria. When gram-negative bacteria were injured by one of several methods, a few species (including E. coli) became sensitive to monensin; this sensitivity was completely reversed in most instances by the inclusion of KCl in the medium. When PMK agar was tested with food and environmental samples, 96% of 535 isolates were gram negative; approximately 68% of colonies from nonselective medium were gram negative. PMK agar was more selective than two other media against gram-positive bacteria and was less inhibitory for gram-negative bacteria. However, with water samples, KCl had an inhibitory effect on gram-negative bacteria, and it should therefore be deleted from monensin-containing medium for water analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y high pressure study.

The process of pressure-induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8-anilino-1-naphthalene sulfonic acid binding fluorescences. Pressure-induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8-anilino-1-naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150-500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8-anilino-1-naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule-like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule-like state to a second molten globule-like state. This two-stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule. A similar three-transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure-induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY.     

Fluorescence spectroscopic studies of Huntington fibroblast membranes.

Using fluorescence spectroscopic methods, we compared the membrane properties of intact fibroblasts from both normal subjects and patients with Huntington disease (HD). Cells were stained with various fluorophores, including 1-anilino-8-naphthalene sulfonic acid (ANS), 2-toluidinyl-6-naphthalene sulfonic acid (TNS), 1,6-diphenyl-1,3,5-hexatriene (DPH), and 6-lauroyl-2-(dimethylamino)-naphthalene (LAURDAN). Using these labeled cells, we measured fluorescence yields and emission maxima (ANS, TNS, and LAURDAN), polarizations (TNS, DPH, and LAURDAN), lifetimes (TNS), and differential polarized lifetimes (DPH). In each instance, comparisons were made between cells from normal and from HD individuals. These cultures were controlled for passage number in culture and for age of donor. We found no significant differences between the HD and the control fibroblasts in experiments using the above-mentioned probes and spectroscopic parameters.     

Antimicrobial properties of diacetyl.

Diacetyl preparations from three commercial sources were found to be essentially similar when tested primarily against a set of 40 cultures, including 10 of lactic acid bacteria, 4 of yeasts, 12 of gram-positive non-lactic acid bacteria, and 14 of gram-negative bacteria. The compound was effective at pH less than or equal to 7.0 and progressively ineffective at pH greater than 7.0. The lactic acid bacteria were essentially unaffected by concentrations between 100 and 350 micrograms/ml over the pH range of 5.0 to 7.0. Of the 12 gram-positive non-lactic acid bacteria, 11 were inhibited by 300 micrograms/ml at pH less than or equal to 7.0. The three yeasts and the 13 gram-negative bacteria that grew at pH 5.5 were inhibited by 200 micrograms/ml. Diacetyl was ineffective against four clostridia under anaerobic conditions. It was lethal for gram-negative bacteria and generally inhibitory for gram-positive bacteria. Nongrowing cells were not affected. The effectiveness of diacetyl was considerably less in brain heart infusion broth, Trypticase soy agar, and cooked-meat medium than in nutrient broth or plate count agar. The antimicrobial activity was antagonized by glucose, acetate, and Tween 80 but not by gluconic acid. As an antimicrobial agent, diacetyl was clearly more effective against gram-negative bacteria, yeasts, and molds than against gram-positive bacteria.     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation     

Measurements of sediment toxicity of autotrophic and heterotrophic picoplankton by epifluorescence microscopy

Sediment toxicity from Toronto (Ontario) and Toledo (Ohio) harbours to autotrophic and heterotrophic picoplankton was evaluated simultaneously using epifluorescent microscopy. The approach is a simple, fast and effective combination of autofluorescence and fluorescence probes - 1-anilino-8-naphthalene sulfonic acid. The procedure is ideally suited for use with sediment slurries since it can differentiate fluorescent biotic material against a background of abiotic sediment particles and detritus.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Antimicrobial activities of some Bacillus spp. strains isolated from the soil

In this study a total of 29 Bacillus species isolated from the soil was analyzed using the agar diffusion method in terms of their general inhibition effects to some test bacteria. It has been found that isolates are effective against gram-positive and gram-negative bacteria whereas their extensive inhibition effect is particularly against gram-positive bacteria. On the other hand, B. cereus M15 strain has an inhibitory effect against both gram-positive and gram-negative bacteria. Furthermore some isolates are more effective against test bacteria when compared to some antibiotics.     

Bacteriology of activated sludge,in particular the filamentous bacteria

Microscopic examination of bulking activated sludge samples showed the presence of a variety of filamentous microorganisms, some of which have not yet been described in the literature. A method was developed to obtain pure cultures of these threaded bacteria. To date, five clearly different groups of filamentous bacteria may be distinguished by the determination of a few morphological and physiological characteristics of the isolates. A variety of sheathed bacteria are included in Group I. Group II includes non-motile, gram-negative, orange- or yellow-pigmented filamentous bacteria. These microorganisms are thought to be related to some species of the genusFlavobacterium. The gram-negative, threaded bacteria of Group III show a more or less distinct gliding movement and form red colonies on rich agar media. These bacteria may apparently be related to species described in the generaMicroscilla andFlexibacter. The filamentous bacteria of Group IV structurally resemble someCyanophyceae, but do not contain photosynthetic pigments. They are gram-positive and non-motile. A number of unknown, non-motile bacteria which stain gram-positive with a variable number of gram-negative autolyzed cells in the filaments, are assigned to Group V. The properties of the isolated bacteria are described briefly and their occurrence in bulking activated sludge is discussed.     

Evidence for a nucleation step in a lipid-protein interaction-kinetics of the incorporation of polymyxin into phosphatidic acid bilayer vesicles

Incubation of 8-anilino-1-naphthalene sulfonic acid with ricin and its isolated A and B polypeptide chains showed an increase in fluorescence at 470 nm. The A chain induced more fluorescence enhancement than either ricin or ricin B chain. The addition of B chain to A chain resulted in decreased fluorescence enhancement which was pH dependent. Sephadex gel filtration showed that A and B chain efficiently reassociated and the reassociation was not dependent on formation of the interchain disulfide bond and could not be prevented by high salt concentration.     

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.     

A protein biosensor for lactate

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.     

Studies on the binding of 1-anilino-8-naphthalene sulfonate to very low density and high density himan serum lipoproteins.

Very low density and high density lipoproteins have been isolated from human plasma and their interaction with 1-anilin0-8-naphthalene sulfonate has been studied under different conditions of pH and added salt. Intrinsic fluorescence of bound 1-anilino-8-naphthalene sulfonate was higher for high density lipoproteins then for very low density lipoproteins, but was unaffected by salt in both systems. Binding of 1-anilino-8-naphthalene sulfonate by both these lipoproteins was saturable and was higher in the presence of added NaCl or CaCl2, Ca2+ having a greater effect than Na+ in enhancing fluorescence. The binding data were analyzed by Scatchard plots; the number of binding sites and the affinity of 1-anilino-8-naphthalene sulfonate for the site increased with increasing salt concentration. Fluorescence pH curves were similar to those published for phospholipids. From these and previous observations it is suggested that the phospholipids probably represent the major binding sites for 1-anilino-8-naphthalene sulfonate.     

Fluorescence study on the interaction of salicylate with rat small intestinal epithelial cells: possible mechanism for the promoting effects of salicylate on drug absorption in vivo

The water-soluble drug, salicylate, was rapidly taken up by rat small intestinal epithelial cells. Salicylate, known to enhance the absorption of poorly absorbable drugs by rectum and small intestine, caused a significant decrease in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a slight increase in the fluorescence polarization of 8-anilino-1-naphthalene sulfonic acid (ANS) in the isolated rat small intestinal epithelial cell suspension. An increase in the membrane fluidity of epithelial cells may possibly contribute to the enhancement of drug absorption by salicylate.     

Relationship between an offensive smell given off from human foot and Staphylococcus epidermidis

The bacteria isolated from foot skins of 17 volunteers by the swab sampling method were mostly gram-positive cocci, which were identified as Staphylococcus epidermidis by the ID-kit SP-18 (Nissui Co., Ltd). After incubation of S. epidermidis on agar plates containing oleic acid and Tween 80 for 24 h at 35 C, the smell noticed was similar to an offensive smell of human pes. However, under the same conditions, the smell of another staphylococcal species was different from that of S. epidermidis. Except for the staphylococcal species, the colonies isolated from the skins were mostly those of yeast (unidentified) and gave off no offensive smell. From these results, it was considered that the smell of human pes might be given off by S. epidermidis, and if this species is inhibited, the smell would also be inhibited. A selective bactericide for gram-positive bacteria, which is a lotion containing deoxycholic acid, was applied to the feet of the 17 volunteers. The experiments showed that the application obviously decreased the counts of colonies of S. epidermidis and inhibited the smell as compared with controls.