Bacterial Viability Tests Research Articles

Effect on Thermal Ink-jet Printing on Bacterial Cells

Ink jetting is a technique with varied applications. The process of bubble/droplet formation of thermal ink jet printers is one that is known to involve a high temperature. This high temperature is however localized and short-lived. Investigations are hereby carried out to evaluate the effect of thermal droplet formation on bacterial cells. Bacterial viability tests were done by colony plate counting and measurement of bacterial absorbance after thermal ink jetting. Bacterial cells were also printed directly onto agar coated glass slides using different templates and incubated. The effect of thermal ink jetting on bacterial cell wall was also conducted by gram staining ink jetted cells. The results obtained indicated that the process of bubble formation did not have any significant effect on bacterial viability as compared to the pipetted versions. Also, bacteria printed directly onto agar gave colonies similar to templates used in printing. Gram staining experiments revealed that the bacterial cells still maintained ability to give a positive gram stain test.

Polyethylene/silver-nanofiber composites: A material for antibacterial films

Silver nanofibers (Ag-Nfbs)~80nm in diameter were synthesized by hydrothermal treatment. The nanofibers (3 and 5wt%) were added in the initial feed together with the catalytic system. Polymerizations in an ethylene atmosphere were performed, yielding PE nanocomposites in situ with 3 and 5wt% content of Ag-Nfbs. The antibacterial effect of the silver-nanofiber composites was evaluated after incubation of Escherichia coli ATCC 25923 for 8h on their surface. Bacterial viability tests showed that the silver-nanofiber composites inhibited the growth of Escherichia coli ATCC 25923 by 88 and 56%. This behavior is attributed to increased silver ions release from the nanocomposite. TEM analysis showed that the antibacterial effect is associated with membrane disruption but not with changes in shape.

Gold nanoparticles mediated coloring of fabrics and leather for antibacterial activity

Metal gold nanoparticles (AuNPs) were synthesized in situ onto leather, silk and cotton fabrics by three different modules, including green, chemical, and a composite of green and chemical synthesis. Green synthesis was employed using Ginkgo biloba Linn leaf powder extract and HAuCl4 with the fabrics, and chemical synthesis was done with KBH4 and HAuCl4. For composite synthesis, G. biloba extract and KBH4 were used to color and embed AuNPs in the fabrics. The colored fabrics were tested for color coordination and fastness properties. To validate the green synthesis of AuNPs, various instrumental techniques were used including UV–Vis spectrophotometry, HR-TEM, FTIR, and XRD. The chemical and composite methods reduce Au+ onto leather, silk and cotton fabrics upon heating, and alkaline conditions are required for bonding to fibers; these conditions are not used in the green synthesis protocol. FE-SEM image revealed the binding nature of the AuNPs to the fabrics. The AuNPs that were synthesized in situ on the fabrics were tested against a skin pathogen, Brevibacterium linens using LIVE/DEAD BacLight Bacterial Viability testing. This study represents an initial route for coloring and bio-functionalization of various fabrics with green technologies, and, accordingly, should open new avenues for innovation in the textile and garment sectors.

Antibacterial effect of kaempferol and (−)-epicatechin on Helicobacter pylori

Phenolic compounds are generated by the secondary metabolism of plants and have been associated with antibacterial properties. Among bacteria affecting human health, Helicobacter pylori has been associated with gastric cancer. There is limited information about the effect of individual or grouped phenolics on H. pylori growth. Previous studies have evaluated the effect of phenolics being part of highly complex food or beverage matrices. The aim of this work was to evaluate the in vitro antibacterial effect of kaempferol and (−)-epicatechin, both individually and combined, on H. pylori in liquid and solid cultures and in co-cultures with AGS human gastric carcinoma cells. Bacterial viability tests were performed in liquid cultures with subsequent CFU/mL counting and in solid cultures by measuring inhibition haloes. Kinetic curves of bacterial growth inhibition in the presence of those phenolics, and the protective effect of (−)-epicatechin on AGS cells against H. pylori infection were characterized. (−)-Epicatechin and kaempferol displayed antibacterial activities, being (−)-epicatechin more effective than kaempferol. After the combined application of both phenols, a synergistic effect of kaempferol plus low but not high doses of (−)-epicatechin was observed. Finally, (−)-epicatechin yielded protection to AGS cells against H. pylori infection. (−)-Epicatechin and kaempferol, both individually and combined, have antibacterial properties and protective effect on H. pylori infection.

Towards effective and stable probiotics.

Probiotics are live microorganisms, generally either lactobacilli or bifidobacteria, which when administered in adequate amounts confer a health benefit to the host [1]. Due to the growing evidence of health benefits associated with their use, probiotics are of increasing interest and represent now a significant growth area in the functional foods industry [2]. However, to be effective, orally administered probiotics should survive preparation of dosage forms and passage through acidic environment of the gastrointestinal tract (GIT). Reaching the intestine, these microorganisms should be able to establish themselves, remain viable and perform their beneficial actions. In this context, oral formulations have to protect probiotic bacteria from gastric acidity and delay their release in the small intestine in order to allow their complete release in the colon. To evaluate effects of starch formulations of lactobacilli on their survival in gastric environment and probiotic properties. Nineteen Lactobacillus strains belonging to the species L. fermentum (14 strains), L. plantarum (4 strains), and L. rhamnosus (1 strain), were isolated from dairy products and probiotics, and were used in this study. Lactobacilli were cultured in de Man, Rogosa, Sharpe (MRS) broth (Merck, Germany) under microaerobic conditions at 37°C.Amylolytic activity of lactobacilli, cultured for 3-5 days on MRS agar supplemented with 1% soluble potato starch (SPS), was determined with iodine reagent (0.01 M I2-KI solution).Loading in starch was performed with L. plantarum 8PA3 bacteria ("Dry lactobacterin", Perm, Russia), which were resuspended to the concentration 1010 cells/mL in 10 mL of 0.85% NaCl solution and added to 90 mL of 2.5% SPS solution. Resulting mixture was frozen at -18°C and then lyophilized (Martin Christ Alpha 1-2 LDplus, Germany).Atomic force microscopy (AFM) images of formulated L. plantarum 8PA3 cells were acquired in air by a Solver P47H atomic force microscope (NT-MDT, Moscow, Russia).Starch swelling and dissolution was studied in simulated colonic fluid (SCF), prepared according to [3] and in distilled water (pH = 6.0) as control. Amylase from Aspergillus oryzae (A8220, Sigma) was added to the solutions to study the influence of amylase. The formulation form was examined visually during 14 h incubation time.Fluorescence microscopy images were obtained with a Leica DM6000B (Germany) fluorescent microscope using Leica FW4000 software.L. plantarum 8PA3 loaded in SPS were placed either in HCl solution (pH 2), or in 2% oxgall bile solution, or in 0.85% NaCl solution. Viability was tested after 2, 4 and 6 h incubation at 37°C by plating diluted aliquots onto MRS agar with subsequent counting of bacterial colony forming units (CFU). In addition, viability was determined using LIVE/DEAD BacLight bacterial viability kit L-7012 (Molecular Probes, Invitrogen) as described elsewhere [4]. Fluorescence in the stained samples was estimated with BD FACS Canto II (USA) flow cytometer or fluorescent microscope.Nitric oxide (NO) production was assessed with DAF-FM DA and DAA fluorescent dyes as described earlier [4]. Each experiment was performed in triplicate. In the present study we studied the probiotic composition comprising of SPS and bacteria L. plantarum 8PA3. We used AFM to confirm effective fixation of the cells to carbohydrate. The compositions were found to swell quickly (~5 min) in aqueous solutions either containing amylase, or not. Tested starch formulations disintegrated during the first 5-10 min of incubation in amylase solutions whereas in amylase-free probes dissolution was less intensive (after ~30 min). Amylolysis of starch excipients was less pronounced in aqueous amylase solution than in SCF, supplemented with amylase. None of 19 studied Lactobacillus strains hydrolyzed SPS when growing on MRS agar supplemented with it. The amount of viable L. plantarum 8PA3 cells formulated with SPS was high and did not change when stored for 6 months at 4°C. The bacterial viability tests also demonstrated that after 6 h treatment with 2% bile or HCl (pH 2) L. plantarum 8PA3 exhibited increased sensitivity (viability 14% and 0.4%, respectively). However, in similar conditions no significant differences were noticed between bacterial viability obtained for formulated with starch and non-formulated bacteria. Furthermore, we showed that loading into SPS had no effect on bacterial production of nitric oxide (NO) - a pluripotent regulatory molecule in human organism. Overall, the results strongly support that formulation with polymeric matrices on the basis of SPS represent an appealing technology of probiotics production. It provides slow release of bacteria in target environment and does not alter their viability and NO biosynthesis. However, SPS excipient does not preserve the bacteria from harsh conditions of upper GIT. Therefore, we conclude that for oral administration the composition should be loaded in acid-resistant capsules.

Antibacterial activity and cell compatibility of TiZrN, TiZrCN, and TiZr-amorphous carbon coatings

A cathodic-arc evaporation system with plasma-enhanced duct equipment was used to deposit TiZrN, TiZrCN, and TiZr/a-C coatings. Reactive gases (N2 and C2H2) activated by the Ti and Zr plasma in the evaporation process was used to deposit the TiZrCN and TiZr/a-C coatings with different C and nitrogen contents. The crystalline structures and bonding states of coatings were analyzed by X-ray diffraction and X-ray photoelectron spectroscopy. The microbial activity of the coatings was evaluated against Staphylococcus aureus (Gram-positive bacteria) and Actinobacillus actinomycetemcomitans (Gram-negative bacteria) by in vitro antibacterial analysis using a fluorescence staining method employing SYTO9 and a bacterial-viability test on an agar plate. The cell compatibility and morphology related to CCD-966SK cell-line human skin fibroblast cells on the coated samples were also determined using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, reverse-transcriptase-polymerase chain reaction, and scanning electron microscopy. The results suggest that the TiZrCN coatings not only possess better antibacterial performance than TiZrN and TiZr/a-C coatings but also maintain good compatibility with human skin fibroblast cells.

Methyl gallate from Galla rhois successfully controls clinical isolates of Salmonella infection in both in vitro and in vivo systems.

Galla rhois is a commonly used traditional medicine for the treatment of pathogenic bacteria in Korea as well as in other parts of Asia. Methyl gallate (MG), a major component of Galla Rhois, exhibits strong antibacterial activity, but its mechanism of action against Salmonella spp. is unclear. In the present study, we investigated the antibacterial actions of MG against Salmonella. The antibacterial activity determined by broth dilution method indicated that the antibacterial activity of MG against Salmonella strains ranged from 3.9 to 125 µg/ml. In vitro bacterial viability test indicated that MG significantly decreased the viability of Salmonella over 40% when combined with ATPase inhibitors. The time-kill curves showed that a combined MG and ATPase inhibitors (DCCD and NaN3) treatment reduced the bacterial counts dramatically after 24 h. Oral administration of MG showed a strong anti-bacterial activity against WS-5 infected BALB/c mice. In contrast to the untreated Salmonella infected control animals, MG treated groups showed no clinical symptoms of the disease, such as lethargy and liver damage. It was observed that MG treatment significantly increased the survival of animals from Salmonella infection, while in untreated groups all animal succumbed to disease by the sixth day post infection. Thus, the present study demonstrates the therapeutic ability of MG against Salmonella infections.

Comparison of LIVE/DEAD® BacLight™ Bacterial Viability Test and alamarBlue® Method for Enumeration of Live and Dead Bacteria for Oral Bacterial Species

LIVE/DEAD® BacLight™ and alamarBlue® are fluorescent materials used for the enumeration of live and dead bacteria. LIVE/DEAD® BacLight™ is generally used for confocal microscopy applications to differentiate live from dead bacteria in a biofilm or planktonic state. AlamarBlue® has also been used widely to assay live and dead bacteria in a planktonic state. Whilst these materials are successfully utilized in experiments to discriminate live from dead bacteria for several species of bacteria, the application of these techniques to oral bacteria is limited to the use of LIVE/DEAD® BacLight™ in biofilm studies. In our present study, we assessed whether these two methods could enumerate live and dead oral bacterial species in a planktonic state. We tested the reagents on Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Enterococcus faecalis and found that only LIVE/DEAD® BacLight™ could differentiate live from dead cells for all five of these oral strains. AlamarBlue® was not effective in this regard for P. gingivalis or A. actinomycetemcomitans. In addition, the differentiation of live and dead bacterial cells by alamarBlue® could not be performed for concentrations lower than 2 × 106 cells/ml. Our data thus indicate that LIVE/DEAD® BacLight™ is a more effective reagent for this analysis.

Anti-bacterial performance of Zirconia coatings on Titanium implants

Bacterial adhesion and colonization are considered to play key roles in the pathogenesis of peri-implant disease, which is an inflammatory process leading the destruction of both soft and hard tissue around a Titanium (Ti) implant. The osseointegration of Ti implants is influenced by their composition and surface treatment. Zirconia (ZrO2) coatings have been shown to improve implant osseointegration in various biomedical fields, including orthopedic devices and dental implants. This study used a twin-gun magnetron sputtering system to deposit ZrO2–Sliver (Ag) and ZrO2–Copper (Cu) coatings on biograde pure-Ti implant materials. The effects of Ag and Cu on the antibacterial properties of the coated Ti samples were investigated using Staphylococcus aureus (S. aureus) and Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans), which are found frequently in implant-associated infections. The in vitro antibacterial analyses employed a fluorescence staining method using SYTO9 and bacterial-viability tests on agar plates. Incorporating Ag and Cu into ZrO2 changed the structure of crystalline ZrO2, and the nanostructures of the resulting ZrO2–Ag and ZrO2–Cu coatings were found to be correlated with the antibacterial performance. The adherence of S. aureus and A. actinomycetemcomitans was less for ZrO2 samples with a small doping of Cu than for uncoated Ti. The ZrO2–Ag coating with an Ag content of 10.6at.%, which showed hydrophobicity, exhibited the lowest bacterial retention and viability of S. aureus and A. actinomycetemcomitans. The results suggest that ZrO2–Ag and ZrO2–Cu coatings improve the antibacterial performance relative to pure-Ti implant materials.

Inactivation of Staphylococcus aureus in Water by a Cold, He/O2 Atmospheric Pressure Plasma Microjet

AbstractA direct‐current, atmospheric pressure, cold plasma microjet (PMJ) sustained in a quasi‐steady gas cavity in liquid was used to inactivate Staphylococcus aureus suspended in distilled water. While helium gas (with 2% O2 as additive) was used as working gas, an effective inactivation (>99%) was achieved in 6 min. The inactivation of bacteria was further verified by surface morphology examination and LIVE/DEAD Baclight bacterial viability test (fluorescence microscopy). The overall pH and temperature of the liquid were monitored during the plasma treatment and were found to be below the critical values for the survival of S. aureus. Hydroxyl radical (•OH) was detected via electron spin resonance (ESR) spectroscopy, and alongside other intermediate reactive species, is attributed to the effective inactivation of S. aureus. End‐on optical emission spectroscopy show strong atomic oxygen emission both in air and in water.magnified image

AI-2/LuxS Is Involved in Increased Biofilm Formation by Streptococcus intermedius in the Presence of Antibiotics

Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P < or = 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.

Water-soluble granules containing Bacillus megaterium for biological control of rice sheath blight: formulation, bacterial viability and efficacy testing

Endospores of B. megaterium were formulated in granule formulations with sodium alginate, lactose and polyvinylpyrrolidone (PVP K-30) by the wet granulation technique. The granule formulation exhibited good physical characteristics, such as high-water solubility and optimal viscosity, that would be suitable for spray application. The bacteria remained viable in the dry granule formulation at 109 c.f.u./g after 24 months storage at room temperature. Under laboratory conditions, aqueous solutions of the formulation showed high activity against mycelial growth of R. solani (99.64 ± 0.14% mycelial inhibition). High viability of the bacterial antagonist on leaf sheath and leaf blade at day 7 after spraying with the formulation was observed (approximately 106 c.f.u./g of plant). Application of an equivalent number of un-formulated endospores resulted in much loss of the bacterial endospores even 1 day after application. In a small pilot field study, an aqueous solution of the formulation (3%w/v) applied by spraying at days 1, 5 and 10 after pathogen inoculation of the rice plants was more effective in suppressing rice sheath blight disease than one application of a fungicide (Iprodione) at day 1. Additionally, rice plants sprayed with the aqueous solution of the granule formulation had higher panicle and whole kernel weights than those of fungicide-treated and control (untreated) plants.

CCL28 Effects on Periodontal Pathogens

Chemokines are small proteins that signal to and attract cells of the immune system; they are vital components in the modulation of immunity and wound healing. A newly described chemokine was reported to have antibacterial and antifungal activity. This chemokine, chemokine (C-C motif) ligand 28 (CCL28; also called mucosae-associated epithelial chemokine), is secreted by mucosal epithelial cells and is found in saliva and in breast milk. The objective of this study was to test whether CCL28 has antibacterial activity against two anaerobic periodontal pathogens: Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans. We used a bacterial viability test, in which two fluorescent dyes are bound differentially to living and killed bacteria. We tested the bacteria at concentrations of 2 x 10(7)/ml, exposing them to CCL28 at concentrations from 0.04 to 10 microM. CCL28 was effective at killing both organisms. After 1 hour of exposure to the chemokine under appropriate oxygen conditions, the percentage of living organisms was reduced significantly for each species. We estimated the 50% effective concentration to be approximately 0.7 microM for P. gingivalis and approximately 2.0 microM for A. actinomycetemcomitans (N = five experiments each). We confirmed these observations using standard bacterial plating methods. Because this chemokine is secreted into the saliva, a reduction in salivary flow (as in xerostomia) may diminish the oral self-defense mechanisms by also reducing the exposure of bacteria to the antibacterial action of CCL28.

Photothermal antimicrobial nanotherapy and nanodiagnostics with self‐assembling carbon nanotube clusters

Unique properties of carbon nanotubes (CNTs) would open new avenues for addressing challenges to realize rapid and sensitive antimicrobial diagnostics and therapy for human pathogens. In this study, new CNTs' capabilities for photothermal (PT) antimicrobial nanotherapy were explored in vitro using Escherichia coli as a model bacterium. Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) were incubated with E. coli K12 strain. CNTs' locations in bacteria and laser-induced thermal and accompanied effects around CNTs were estimated with TEM and PT microscopy, respectively. Multi-pulse lasers at 532 and 1064 nm with 12-ns pulse duration were used for irradiating sample mixtures at different laser fluences. Cell viability was evaluated using a bacterial viability test kit and epi-fluorescence microscopy. This study revealed CNTs' high binding affinity to bacteria, their capability to self-assemble as clusters at bacteria surfaces, and their inherent near-infrared (NIR) laser responsiveness. Cell viability was affected neither by CNTs alone nor by NIR irradiations alone. Notable changes in bacteria viability, caused by local thermal and accompanied bubble-formation phenomena, were observed starting at laser fluences of 0.1-0.5 J/cm(2) with complete bacteria disintegration at 2-3 J/cm(2) at both wavelengths. Furthermore, ethanol in reaction mixtures significantly (more than one order) enhanced bubble formation phenomena. This first application of laser-activated CNTs as PT contrast antimicrobial agents demonstrated its great potential to cause irreparable damages to disease-causing pathogens as well as to detect the pathogens at single bacterium level. This unique integration of laser and nanotechnology may also be used for drinking water treatment, food processing, disinfection of medical instrumentation, and purification of grafts and implants. Furthermore, the significant ethanol-induced enhancement of bubble formation provides another unique possibility to improve the efficiency of selective nanophotothermolysis for treating cancers, wounds, and vascular legions.

The Antimicrobial Effect of Silver Ion Impregnation into Endodontic Sealer against Streptococcus mutans

Pulpal and periradicular diseases are primarily caused by bacterial invasion of the root canal system as a result of caries progression. The presence of residual bacteria at the time of root canal completion (obturation) is associated with significantly higher rate of treatment failure. Re-infection of obturated root canals can be potentially prevented by enhancing the antibacterial activities of root canal obturation materials. We evaluated, in an in vitro model, the antimicrobial efficacy of silver ions added to a common endodontic sealer. For that purpose we performed growth inhibition studies and bacterial viability tests. We measured the zone of inhibition, optical density and performed confocal laser scanning microscopy. Our results show that the silver ions enhance the antimicrobial activity of the root canal sealer against Streptococcus mutans. This study approach may hold promise for studying other biologically based therapies and therefore increasing the success rate of routine orthograde root canal treatment.

Killing of Periodontopathogenic Bacteria by Photodynamic Therapy

The aim of this study was to evaluate a new approach for killing periodontopathogenic bacteria using photodynamic therapy (PDT). In this study, we investigated the photosensitizers chlorin e6, BLC 1010, and BLC 1014 by three different methods for their effect in PDT on the viability of periodontopathogenic bacterial species. The methods included examination of inhibition zones on agar plates, determination of colony-forming units (CFU), and the use of a bacterial viability kit. Using the CFU method, we were able to demonstrate that the anaerobic bacteria Porphyromonas gingivalis, Fusobacterium nucleatum, and Capnocytophaga gingivalis can be photoinactivated completely by illumination with an intensity of 5.3 J/cm2 in the presence of 10 microM chlorin e6 and 10 microM BLC 1010. With the photosensitizers chlorin e6 and BLC 1010, we were able to induce zones of inhibition on agar plates. BLC 1014 failed to produce a zone of inhibition. The results of the bacterial viability test also showed that the photosensitizer BLC 1014 provides the lowest photodynamic effect in comparison to the others. The data collected to date suggest that photodynamic therapy with chlorin e6 and BLC 1010 is advantageous for suppressing periodontopathogenic bacteria.

Mechanisms of microwave irradiation involved in the destruction of fecal coliforms from biosolids

Microwaves have been found to be effective in destructing pathogens in sewage sludge (biosolids) (75th Annual Water Environment Federation Conference, Chicago, September 29–October 2, 2002; Third World Water Congress, International Water Association, Melbourne, Australia, April 7–12, 2002). Mechanisms and roles of microwaves on fecal coliform destruction were investigated using bacterial viability tests, electron transport system (ETS) and β-galactosidase activity assays, gel electrophoresis, and genomic deoxyribonucleic acid (DNA) optical density (OD) measurements with fecal coliforms isolated from biosolids. Bacterial viability tests demonstrated cell membrane damage as microwave irradiation intensity and temperature increased. Above 60±3°C, viable cells were rarely found when pure fecal coliforms were irradiated with microwaves. ETS and β-galactosidase activity assays revealed increased activity for externally heated samples due to fecal coliform growth but decreased activity for microwave-irradiated samples as temperature was increased from 20°C to 57°C, indicating other destruction mechanisms besides heating. Between 57°C and 68°C, microwave irradiation led to a more rapid decrease in activity than external heating by convection. Above 68°C, bacterial activity almost ceased for both pretreatments. DNA bands in gel electrophoresis tests and OD of genomic DNA decreased more rapidly for microwave-irradiated samples than for externally heated samples, implying that microwaves disrupted DNA in fecal coliform cells at lower temperatures than external heating. Microwave irradiation of sludge appears to be a viable and economical method of destructing pathogens and generating environmentally safe sludge.

Kinetics of Nitrosomonas europaea INACTIVATION by chloramine

The effect of pH on the inactivation of Nitrosomonas europaea by chloramines was tested for a range of chloramine concentrations. Cell viability was assessed using the most probable number (MPN) technique and a culture‐independent bacterial viability test. For pH 7 to 9, N. europaea inactivation followed Chick‐Watson kinetics with n = 1 and k ranging from 2.5 × 10–03 L/(mgCl2·min) at pH 7 to 2.4 × 10–04 L/(mgCl2·min) at pH 9 in the bacterial viability–based tests. For the MPN‐based tests, k was approximately 1,000‐fold higher. Although CT99 values from the MPN‐based experiments agreed with CT99 reported elsewhere for ammonia‐oxidizing bacteria (AOB), the lower inactivation rates estimated from the bacterial viability tests were more consistent with AOB persistence in distribution systems, suggesting that MPN may overestimate chloramine biocide effectiveness. Finally, the counteracting effect of pH on N. europaea inactivation and chloramine decay indicates that pH control is likely to have a site‐specific effect on nitrification.

Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.

Marine algae that display anti-tumorigenic activity against Agrobacterium tumefaciens

Thirty-five extracts representing different seasonal growths of 17 marine algal species collected from the Alexandria coast were tested for anti-tumorigenic activity against Agrobacterium tumefaciens galls on potato discs. Eleven extracts (nine species) displayed > 20% inhibition of tumor initiation, with three of these ( Codium tomentosum, winter; Jania rubens, summer; Padina pavonia, winter) displaying relatively high activity. Bacterial viability tests showed that the inhibitory effects were directly due to anti-tumorigenesis rather than an indirect result of anti-bacterial activity.