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PubMed1981

Comparison of two direct-count methods for determining metabolizing bacteria in freshwater.

Planktonic bacteria collected from several freshwater environments and cultured bacteria were used to compare two methods for determining the numbers of metabolizing bacteria. The methods used were (i) reduction of 2-(rho-iodophenyl)-3-(rho-nitrophenyl)-5-phenyl tetrazolium chloride 2-(rho-iodophenyl)-3-(rho-nitrophenyl)-5-phenyl to tetrazolium chloride-formazan and (ii) elongation of cells by using yeast extract and nalidixic acid. No statistically significant difference was found between methods in determining metabolizing bacteria, although significant differences (P < 0.05) were found when comparing numbers of total bacteria. A combination of the two methods yielded significant changes, both positive and negative, in the numbers of metabolizing bacteria.

Journal Article↗

PubMed1982

Tetrazolium reduction-malachite green method for assessing the viability of filamentous bacteria in activated sludge.

A method was developed to assess the activity of filamentous bacteria in activated sludge. It involves the incubation of activated sludge with 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride followed by staining with malachite green. Both cells and 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride-formazan crystals can be observed in prepared specimens by using bright-field microscopy. This procedure allowed us to distinguish between inactive and actively metabolizing filaments after chlorine application to control the bulking of activated sludge.

Journal Article↗

PubMed1983

Starvation-survival processes of a marine Vibrio.

Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated.

Journal Article↗

PubMed1988

A simple, rapid method for enumerating total viable and metabolically active bacteria in groundwater.

We report here a new staining procedure which uses both the enzymatic dehydrogenation of 2-(p-iodophenyl)-3-p-(nitrophenyl)-5 phenyltetrazolium chloride to a pink intracellular formazan and the DNA-specific fluorochrome 4',6'-diamidino-2-phenylindole. Application of this staining procedure to cells concentrated on filters and then transferred to microscope slides by the filter-transfer-freeze technique has proven valuable for statistically accurate enumeration of the total viable and metabolically active cells in groundwaters.

Journal Article↗

PubMed1989

Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water.

Some effects of visible light on the survival of Escherichia coli in waters of the Butrón river were studied by comparing illuminated and nonilluminated systems. The following count methods were used: CFU on a selective medium (eosin-methylene blue agar), CFU on a medium of recuperation (Trypticase soy agar with yeast extract and glucose), number of metabolically active cells by reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, and total number of E. coli cells as determined by the acridine orange direct-count method. In the illuminated systems, decreases in CFU of E. coli and in the number of metabolically active cells were observed. However, no decline of the total number of E. coli cells was observed. By count methods, different stages of progressive dormancy of E. coli cells were determined to exist in illuminated systems. Culturable and recoverable cells were defined as viable cells, and metabolically active cells and morphologically intact cells were defined as somnicells. Indirect activity measurements were also done by using [14C]glucose. In illuminated systems, a decrease of glucose uptake by E. coli cells was observed throughout the experiments. The assimilated fraction of [14C]glucose decreased faster than the respired fraction in illuminated systems. The percentage of respired [14C]glucose (14CO2 production) with respect to the total glucose uptake increased throughout the experiments, and the percentage of assimilated glucose decreased. Therefore, the visible light was also responsible for an additional inhibition of biosynthetic processes.

Colony Count, Microbial↗

PubMed1991

Contemporaneous N(2) Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii.

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N(2) fixation associated with mats. The ability to contemporaneously conduct O(2)-sensitive N(2) fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O(2) evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N(2) fixation (nitrogenase activity), indicating potential inhibition of N(2) fixation by O(2) production. However, some degree of light-mediated N(2) fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of CO(2) fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) CO(2) fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N(2) fixation during illumination, with N(2) fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N(2) fixation.

Journal Article↗

PubMed1991

Development of the BIOLOG substrate utilization system for identification of Legionella spp.

The genus Legionella consists of 51 serogroups comprising 34 species. Biochemical reactions and cell wall fatty acid and quinone analyses may confirm that an isolate is a Legionella sp. and indicate to which species it belongs, but DNA hybridization studies have been necessary for a definitive identification. Recently, the commercially available BIOLOG identification system has offered a standardized, easily reproducible system of substrate metabolism by bacteria resuspended in multiwell plates. A tetrazolium dye acts as an electron acceptor during the oxidation of the wide range of substrates and forms an irreversible, highly colored formazan when reduced. The 95 substrate wells are read rapidly with a conventional plate reader, and the results are downloaded for comparison with a computer data base, allowing quick identification. The BIOLOG system's ability to test more diverse classes of substrates, including amino acids, peptides, carboxylic acids, and carbohydrates, was used in this study to establish a new data base and identify the asaccharolytic Legionella spp. In particular, Legionella pneumophila behaved as a microaerophile, and the fastest, most diverse metabolic activities occurred after the development of a low-oxygen incubation environment. Alternatively, bacteria could be successfully incubated in air when their concentration was double that recommended by the manufacturer. Similar results were obtained by using either Page's amoebal saline or distilled water as the resuspending and incubation medium. Type strains did not cross-identify with any of the strains already in the manufacturer's data base. The results indicate that this modified system has value in being able to identify Legionella isolates to the species level.

Bacterial Typing Techniques↗

PubMed1993

Dormancy in Stationary-Phase Cultures of Micrococcus luteus: Flow Cytometric Analysis of Starvation and Resuscitation.

Cultures of the copiotrophic bacterium Micrococcus luteus were stored in spent growth medium for an extended period of time following batch culture. After an initial decrease, the total cell counts remained constant at approximately 60 to 70% of the counts at the beginning of storage. The level of viability, as judged by plate counts, decreased to less than 0.05%, while respiration and the ability to accumulate the lipophilic cation rhodamine 123 decreased to undetectable levels. However, using penicillin pretreatment (to remove viable cells) and flow cytometry and by monitoring both the total and viable counts, we found that at least 50% of the cells in populations of 75-day-old cultures were not dead but were dormant. Resuscitation in liquid medium was accompanied by the appearance of a population of larger cells, which could accumulate rhodamine 123 and reduce the dye 5-cyano-2,3-ditolyl tetrazolium chloride to a fluorescent formazan, while a similar fraction of the population was converted to colony-forming, viable cells. We surmise that dormancy may be far more common than death in starving microbial cultures.

Journal Article↗

PubMed1993

Survival of denitrifiers in nitrate-free, anaerobic environments.

Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO(3)-free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 x 10 and 3.1 x 10 cells cm in deep sediments from a river and lake, respectively, and 5.3 x 10 cells cm in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predominant isolates that would grow on anaerobic medium with NO(3). All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of Pseudomonas and Alcaligenes. No detectable growth was observed when these strains were incubated with electron acceptors other than NO(3) or O(2). When representative isolates were added to sterile, O(2)- and NO(3)-free porewater from their original locations at their natural densities (10 cells cm), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O(2) or NO(3)), C-labeled products, including cell biomass, CO(2), and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O(2) or NO(3) and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.

Journal Article↗

PubMed1993

Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water.

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-microns-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindole. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces.

Nisin is an antimicrobial peptide proven to be an effective inhibitor of gram-positive bacteria. It is known that nisin can adsorb to various surfaces and still retain much of its original activity (M. A. Daeschel, J. McGuire, and H. Al-Makhlafi, J. Food Prot. 55:731-735, 1992). In this study, nisin films were allowed to form on silanized silica surfaces and then exposed to medium containing Listeria monocytogenes. Representative areas were selected from each surface, and images of resident listeriae were obtained at 4-h intervals for 12 h. During this time, cells on surfaces that had been in contact with a high concentration of nisin (1.0 mg/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces treated with a lower concentration of nisin (0.1 mg/ml) had a smaller degree of inhibition. In contrast, both protein-free surfaces and those with films of heat-inactivated nisin allowed attached L. monocytogenes cells to grow and reproduce. These studies, when repeated with a nisin-resistant strain of L. monocytogenes, resulted in no inhibition of growth on surfaces with adsorbed nisin. The bactericidal effect of adsorbed nisin was also studied with iodonitrotetrazolium violet, a tetrazolium salt, which is reduced to a red formazan crystal by viable bacteria. Crystals were visible in 95% of the cells adhered to control surfaces but were present in less than 20% of the cells on surfaces with adsorbed nisin. These data indicate that adsorbed nisin may have potential for use as a food grade antimicrobial agent on food contact surfaces.

Adsorption↗

PubMed1995

A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water.

Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed. We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria. Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium. A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter. The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA. Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water. Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E. coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC. Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997). Differences in injury did not appear to affect CTC reduction. The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.

Culture Media↗

PubMed2003

Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay.

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na+ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na+ and K+, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na+ and K+ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.

Bacteriolysis↗

PubMed1973

Tetrazolium reduction as a measure of metabolic activity for glass-adherent Mycoplasma pneumoniae.

The reduction of tetrazolium was used to assay the metabolic capability of developing Mycoplasma pneumoniae cultures on glass. Generally, the amount of tetrazolium reduced correlates with the amount of growth as measured by protein. Until a culture enters the late phase of the growth cycle, the drop in pH of culture medium provides similar information. In this last stage of growth, protein appears to be leveling. The pH continues to fall, but tetrazolium reducing activity decreases. Thus, considering the entire M. pneumoniae growth cycle, formazan production is a more reliable measure of metabolic capability of the organisms than either protein or pH. The reduction of tetrazolium provides a quantitative means of assessing enzymatic activities of glass-adherent M. pneumoniae.

Azo Compounds↗

PubMed1963

INHIBITORY ACTION OF PHAGE K ON STAPHYLOCOCCAL DEHYDROGENASES. I. EFFECT ON VARIOUS STRAINS OF STAPHYLOCOCCUS AUREUS, INCLUDING MEMBERS OF THE PHAGE-TYPING SERIES.

Ralston, D. J. (University of California, Berkeley) and B. S. Baer. Inhibitory action of phage K on staphylococcal dehydrogenases. I. Effect on various strains of Staphylococcus aureus, including members of the phage-typing series. J. Bacteriol. 86:666-672. 1963.-The polyvalent phage K was found to depress the dehydrogenase activities of a large number of Staphylococcus aureus strains, as measured by the reduction of triphenyl tetrazolium chloride to the insoluble red formazan. The inhibition occurred immediately after the absorption of a multiplicity of phage particles, and was independent of the infectibility of the strains and of the killing ability of the phage. It appeared to be closely associated with the phenomenon of sensitization-a change in the cell surface which increased the susceptibility of the wall to digestion by soluble staphylococcal lysins and simultaneously abolished the capacity to synthesize phage. The inhibitory effect occurred both in nutrient media and in a nongrowth glucose - phosphate buffer supplemented with cysteine.

Bacteriophage Typing↗

PubMed1994

An automated enzymatic method for measurement of D-arabinitol, a metabolite of pathogenic Candida species.

An automated enzymatic method was developed for the measurement of D-arabinitol in human serum. The assay is based on a novel, highly specific D-arabinitol dehydrogenase from Candida tropicalis. This enzyme catalyzes the oxidation of D-arabinitol to D-ribulose and the concomitant reduction of NAD+ to NADH. The NADH produced is used in a second reaction to reduce p-iodonitrotetrazolium violet (INT) to INT-formazan, which is measured spectrophotometrically. The entire reaction sequence can be performed automatically on a COBAS MIRA-S clinical chemistry analyzer (Roche Diagnostic Systems, Inc., Montclair, N.J.). Replicate analyses of human sera supplemented with D-arabinitol over a concentration range of 0 to 40 microM demonstrated that the pentitol could be measured with an accuracy of +/- 7% and a precision (standard deviation) of +/- 0.4 microM. Serum D-arabinitol measurements correlated with those determined by gas chromatography (r = 0.94). The enzymatic method is unaffected by L-arabinitol, D-mannitol, or other polyols commonly found in human serum. Any of 17 therapeutic drugs potentially present in serum did not significantly influence assay performance. Data illustrating the application of the assay in patients for possible diagnosis of invasive candidiasis and the monitoring of therapeutic intervention are presented. The automated assay described here was developed to facilitate the investigation of D-arabinitol as a serum marker for invasive Candida infections.

Agranulocytosis↗

PubMed2001

Biofilm formation by Candida dubliniensis.

Candida dubliniensis is an opportunistic yeast closely related to Candida albicans that has been recently implicated in oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Most manifestations of candidiasis are associated with biofilm formation, with cells in biofilms displaying properties dramatically different from free-living cells grown under normal laboratory conditions. Here, we report on the development of in vitro models of C. dubliniensis biofilms on the surfaces of biomaterials (polystyrene and acrylic) and on the characteristics associated with biofilm formation by this newly described species. Time course analysis using a formazan salt reduction assay to monitor metabolic activities of cells within the biofilm, together with microscopy studies, revealed that biofilm formation by C. dubliniensis occurred after initial focal adherence, followed by growth, proliferation, and maturation over 24 to 48 h. Serum and saliva preconditioning films enhanced the initial attachment of C. dubliniensis and subsequent biofilm formation. Scanning electron microscopy and confocal scanning laser microscopy were used to further characterize C. dubliniensis biofilms. Mature C. dubliniensis biofilms consisted of a dense network of yeasts cells and hyphal elements embedded within exopolymeric material. C. dubliniensis biofilms displayed spatial heterogeneity and an architecture showing microcolonies with ramifying water channels. Antifungal susceptibility testing demonstrated the increased resistance of sessile C. dubliniensis cells, including the type strain and eight different clinical isolates, against fluconazole and amphotericin B compared to their planktonic counterparts. C. dubliniensis biofilm formation may allow this species to maintain its ecological niche as a commensal and during infection with important clinical repercussions.

AIDS-Related Opportunistic Infections↗

PubMed1976

Lactate dehydrogenase isoenzyme pattern in the eye muscles. Deviation in myopia.

The lactate dehydrogenase isoenzyme patterns in 46 extraocular eye muscle samples removed at surgery for squint were determined by acrylamide-gel electrophoresis and reduction of NAD coupled with formazan reaction. Muscle type subunits predominated in the isoenzymes of the medial and lateral rectus muscles of emmetropic and hypermetropic eyes, whereas heart type subunits predominated in those of myopic eyes.

Adolescent↗