Colony-forming Unit Method Research Articles

Mast cells kill Candida albicans in the extracellular environment but spare ingested fungi from death.

Mast cells (MCs) reside in tissues that are common targets of Candida spp. infections, and can exert bactericidal activity, but little is known about their fungicidal activity. MCs purified from rat peritoneum (RPMC) and a clinical isolate of C. albicans, were employed. Ingestion was evaluated by flow cytometry (FACS) and optical microscopy. The killing activity was assayed by FACS analysis and by colony forming unit method. RPMC degranulation was evaluated by β-hexosaminidase assay and phosphatidylserine externalization by FACS. Phagocytosing RPMC were also analyzed by transmission electron microscopy. Herein, we show that the killing of C. albicans by RPMC takes place in the extracellular environment, very likely through secreted granular components. Ultrastructural analysis of the ingestion process revealed an unusual RPMC-C. albicans interaction that could allow fungal survival. Our findings indicate that MCs have a positive role in the defense mechanism against Candida infections and should be included among the cell types involved in host-defense against this pathogen.

Effect of estradiol on planktonic growth, coaggregation, and biofilm formation of the Prevotella intermedia group bacteria

Alterations in the quantity and quality of biofilms at gingival margin are considered to play a role in the initiation and development of pregnancy-related gingivitis. Prevotella intermedia sensu lato is able to consume estradiol, the major sex hormone secreted during pregnancy, in the absence of vitamin K. The aim of the study was to examine the effect of estradiol on the planktonic growth, coaggregation, polysaccharide production, and biofilm formation of the P. intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, and Prevotella pallens. In all experiments, the type strain (ATCC) and a clinical strain (AHN) of P. intermedia, P. nigrescens, and P. pallens were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol. Planktonic growth was assessed by means of the colony forming unit method, while coaggregation and biofilm formation were assessed by spectrophotometric methods. In the determination of protein and polysaccharide levels, the Bradford and phenol–sulfuric acid methods were used, respectively. P. pallens AHN 9283 and P. nigrescens ATCC 33563 increased their numbers at planktonic stage with increasing estradiol concentrations. In 48-h biofilm tests, elevated protein levels were found for both strains of P. intermedia, and the strains P. nigrescens ATCC 33563 and P. pallens AHN 9283 in the presence of estradiol. The P. intermedia strains also increased the levels of polysaccharide formation in the biofilm. Coaggregation of the P. intermedia group organisms with Fusobacterium nucleatum was enhanced only in P. intermedia AHN 8290. In conclusion, our in vitro experiments indicate that estradiol regulates planktonic growth, coaggregation, polysaccharide production, and biofilm formation characteristics of P. intermedia, P. nigrescens, and P. pallens differently. These results may, at least partly, explain the differences seen in their contribution to the pathogenesis of pregnancy-related gingivitis.

Effect of Plasma Processing and Organosilane Modifications of Polyethylene onAeromonas hydrophilaBiofilm Formation

The aim of our research was to study how the modifications of polyethylene—a material commonly used in medicine and water industry—influence bacterial cell attachment and biofilm formation. The native surface was activated and modified using two-step process consisting in the activation of native surface with a H2O vapor plasma followed by its treatment with various organosilanes, namely, [3(tertbutylamine-2hydroxy) propyloxypropyl] diethoxymethylsilane, 1H,1H,2H,2H-perfluorooctylmethyldimethoxysilane, dimethoxydimethylsilane, and isobutylmethyldimethoxysilane. The effect of polyethylene modification after chemical treatment was analyzed using surface tension measurement. The adhesive properties of Aeromonas hydrophila LOCK0968 were studied in water with a low concentration of organic compounds, using luminometric and microscopic methods, and the viability of the adhered bacterial cells was evaluated using the colony forming units method. After two-week incubation the chemically modified materials exhibited better antiadhesive and antibacterial characteristics in comparison to the native surface. Among the examined modifying agents, dimethoxydimethylsilane showed the best desired properties.

Antimicrobial Efficacy Tests of Multipurpose Contact Lens Care Solutions Simulating Poor Contact Lens Hygiene Behaviors

The aim of this study was to simulate the biofilm formation in contact lens (CL) case under poor hygiene behaviors; antimicrobial efficacies of multipurpose solutions (MPSs) against biofilm on the lens case were evaluated. Five MPSs (Epica Cold, Complete 10 min, ReNu MultiPlus, SoftOne Mois, and OPTI-FREE Plus) were tested. Lens cases containing ACUVUE2 were inoculated with 1×10, 10, or 10 colony-forming units (CFUs) of Staphylococcus epidermidis (SE). Each lens case was treated with 1 MPS for 4 hrs followed by the estimation of the number of SE by the CFU method. Disinfection efficacies of MPSs against SE biofilm were evaluated by biomicroscopy with safranin staining and scanning electron microscopy. Lens cases, inoculated with 1×10 CFU, were disinfected by all MPSs. Epica Cold, Complete 10 min, ReNu MultiPlus, and OPTI-FREE showed almost a 2-log reduction of the CFU, whereas SoftOne Mois effect was almost a 1 log reduction, significantly lower than other MPSs (P<0.05). No biofilm formations were observed in Epica Cold, Complete 10 min, ReNu MultiPlus, and OPTI-FREE Plus-treated groups unlike significant biofilm formation in the SoftOne Mois-treated group (P<0.01). Greater efforts to educate patients regarding compliant lens care behavior are needed to reduce the incidence of CL-associated microbial keratitis.

Determine the Potency of BCG Vaccines by Flow Cytometer

ABSTRACTThe Bacille Calmette-Guerin (BCG) vaccine is a live attenuated vaccine prepared from a strain of Mycobacterium bovis. The potency of this vaccine is dependent on the amount of viable cells. The conventional method to determine the potency of the BCG vaccine is to measure the number of colony forming units (CFU) produced after the vaccine has been reconstituted in normal saline and the cells cultured on Löwenstein-Jensen (LJ) medium. However, as M. bovis is a slow growing organism, it needs to grow at least four weeks to be detected in the potency assay. The results of the potency assay could also vary due to manual counting. Therefore, in order to increase the assay efficiency and decrease the variation of the results, we developed an alternative method by using flow cytometer combined with cell counter (viable counts calculated from flow cytometer and counter, VFC) to determine the potency of BCG vaccines. To further test the accuracy of the assay we developed, the viable counts of ten batches of BCG vaccine determined by the VFC assay and conventional CFU method were compared. Wilcoxon statistical analysis showed that there were no statistically significant differences between the VFC assay and the conventional CFU method. Moreover, the results of the VFC assay are all read out from instruments, which could reduce the variation from different operators in order to overcome the limitation of the conventional CFU method. In summary, the results demonstrated that the potency of BCG vaccines could be determined by the VFC assay within four hours instead of four weeks cultivation by the conventional CFU method. On the whole, compared to the conventional CFU method, the VFC assay is a rapid, efficient and reliable method to determine the potency of BCG vaccines.

Photoinactivation effects of hematoporphyrin monomethyl ether on Gram-positive and -negative bacteria detected by atomic force microscopy

The photodynamic antimicrobial chemotherapy as a promising approach for efficiently killing pathogenic microbes is attracting increasing interest. In this study, the cytotoxic and phototoxic effects of hematoporphyrin monomethyl ether (HMME) on the Gram-positive and Gram-negative bacteria were investigated. The cell viability was assessed by colony-forming unit method, and the results indicated that there was no significant cytotoxicity but high phototoxicity in the examined concentrations. Notably, the Gram-positive bacteria were more sensitive to HMME in phototoxicity. Simultaneously, an atomic force microscope (AFM) was used to detect the changes in morphological and nanomechanical properties of bacteria before and after HMME treatment. AFM images indicate that upon photoinactivation, the bacterial surface changed from a smooth, homogeneous architecture to a heterogenous, crackled morphology. The force spectroscopy measurements reveal that the cell wall became less rigid and the Young's modulus decreased about 50%, whereas the tip-cell-surface adhesion forces increased significantly compared to those of native cells. It was speculated that the photodynamic effects of HMME induced the changes in the chemical composition of the outer membrane and exposure of some proteins inside the envelope. AFM can be utilized as a powerful and sensitive method for studying the interaction between bacteria and drugs.

Wood Colour Test of Fisherman’s Boat on Microorganism Linkage in Kangean

Most of Indonesian fishermen have been using wooden boat. The boat has been used by fishermen in the north shore of East Java are made from Teak and Silk cotton tree. The leakage indication had not been shown on its linkage but on the boat caused by a microor-ganism called biofouling. The research used Colony For-ming Unit method (CFU) and Saburoud Dextrose Agar (SDA) as a fungus counting medium and Nutient Agar (NA) as a bacteria counting medium. Each medium had been adjusted to the microorganism. The result shows that the wood and the paint which were used to build the boat influenced the type of microorganism, fungus, and bac-teria on the boat. Bacteria preferred to stick on the yellow boat, and fungus preferred to stick on the green boat. The microorganism had been identified on the red Nyamplung tree, Kosambi tree, Jackfruit tree, Kamfer tree, and Teak tree are Micrococcus sp, Bacillus sp, and Pseudomonas sp, and all of those are bacteria. The other microorganism had been identified were Aspergillus niger, Aspergillus oryzae, Penicillium sp, Candida sp, Rhodotorulla sp, Saccharomyces sp, Verticillium sp, Paecilomyces sp, Monillia sp, and Altenaria sp, and all of those are fungus.

Role of connexin-43 hemichannels in the pathogenesis of Yersinia enterocolitica

Connexin (Cx) channels are sites of cytoplasmic communication between contacting cells. Evidence indicates that the opening of hemichannels occurs under both physiological and pathological conditions. In this paper, the involvement of Cx-43 hemichannels is demonstrated in the pathogenesis of Yersinia. Parental HeLa cells and transfected HeLa cells stably expressing Cx-43 (HCx43) were infected with Yersiniaenterocolitica, and bacterial uptake was measured by the colony-forming unit method. Bacterial uptake was higher in HCx43 cells than in parental cells and was inhibited by the Cx channel blocker, 18-alpha-glycyrrhetinic acid (AGA). The inhibitory effect of AGA was more pronounced on the Y. enterocolitica uptake by HCx43 cells than by parental cells. The ability of HCx43 cells to incorporate the permeable fluorescent tracer Lucifer Yellow (LY) was assessed. Dye incorporation was inhibited by AGA, whereas Y. enterocolitica infection of HCx43 cells increased LY incorporation. Western blotting analysis demonstrated that Y. enterocolitica infection of HCx43 cells induced tyrosine phosphorylation of Cx-43, thus supporting a critical role for Cx-43 in the strategies exploited by bacterial pathogens to invade non-phagocytic cells.

Evidence that the p38 MAP kinase pathway is dysregulated in HLA–B27–expressing human monocytic cells: Correlation with HLA–B27 misfolding

To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding. U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wild-type HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy. Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F). Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

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.

Enhanced intracellular replication of Salmonella enteritidis in HLA-B27-expressing human monocytic cells: dependency on glutamic acid at position 45 in the B pocket of HLA-B27.

To reveal the cause of the impaired elimination of Salmonella enteritidis in HLA-B27-transfected human monocytic cells and to study whether the B pocket of HLA-B27 contributes to these modulatory effects. Stable U937 cell transfectants expressing HLA-A2, B27, or different forms of B27 with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vector (pSV2neo) alone. Cells were differentiated, infected with S enteritidis, and the number of live intracellular S enteritidis organisms was determined using the colony-forming unit method. To visualize intracellular S enteritidis, the bacteria were transformed with green fluorescent protein (GFP), and studied by confocal microscopy. Cells expressing wild-type HLA-B27 were more permissive of intracellular replication of S enteritidis compared with mock-transfected or A2-transfected controls. Cells expressing B27 with an altered B pocket composition having either 6 amino acid substitutions (B27.A2B; substitutions H9F, T24A, E45M, I66K, C67V, and K70H) or a single substitution (B27.E45M) were no longer permissive of S enteritidis replication. In contrast, cells expressing B27 with the single substitution of F for H at position 9 (B27.H9F) retained their permissiveness. Studies using GFP-transformed S enteritidis confirmed that the increase in the amount of intracellular bacteria in B27-expressing cells was due to replication of the bacteria. Our data indicate that HLA-B27 expression modulates the host-microbe interaction that results in an impaired capacity of monocytes to resist intracellular replication of S enteritidis. The phenotype is dependent on glutamic acid at position 45 in the B pocket and, thus, may be due to properties of the B27 heavy chain that are related to this residue. The ability of HLA-B27 to confer susceptibility to Salmonella-triggered reactive arthritis may occur, at least in part, through these modulatory effects.

Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method

1 A biochemical method, based on dehydrogenase activity (DHA) measurement, has been developed as an alternative to colony forming unit (CFU) enumeration, for assessing the viability of fungal spores. In viable cells, a tetrazolium salt (MTT) is reduced to a coloured formazan (MTTf) by cellular dehydrogenase enzymes. From the colorimetric assay developed by Mosmann for mammalian cells, the procedure has been adapted and optimised using P. digitatum spores as a model. Propan-2-ol has been selected as the best solvent to extract the MTTf from the spores. The sensitivity of the method has been considerably increased by determining the optimal conditions of incubation for the MTT reduction by spores: temperature at 50°C and pH 8. Using the assay, a correlation has been established between DHA and the number of viable spores as measured by the CFU method (10 5 CFU = 1.14 DHA -26; r 2 = 0.94). This standardised procedure allows the viability of P. digitatum spores to be estimated with accuracy and reliability. Moreover, the extension of this assay to other strains has been demonstrated ( Aspergillus niger and Metarhizium flavoviride).

Microorganisms and plant of Autonomous Biological Systems (ABS) samples.

Distribution of microorganisms and cellular structure of an Autonomous Biological Systems (ABS) were studied with a special attention to the effect of space environments. Viable cell densities measured by the direct fluorescence microscopic method were in the order of 10(5) cells/ml for fractions 1 (upper suspension) and 2 (lower suspension), and 10(6) cells/ml for fraction 3 (sediments). These values were 10 to 100 times larger than the values obtained by the classical colony forming unit method. No difference between flight and ground samples was observed in the vertical distribution of viable microorganisms when fractionation and analysis were carried out after recovery. Intracellular distribution of chloroplasts in higher green plants, Ceratophyllum demersum, of flight samples was disturbed after 10 days of flight (24hrs/day light on). After 4 months of flight (Mir/STS-79/81) with 24 hrs light on, Ceratophyllum demersum was completely disintegrated. On the other hand, in the second 4-months-flight experiment with 16 hrs/day light on, Ceratophyllum demersum was only slightly deteriorated.

The use of epi-fluorescence to determine the viability of Saccharomyces cerevisiae subjected to osmotic shifts

The viability of yeast cells subjected to osmotic shifts was determined using an epi-fluorescence method in conjunction with an image analysis system. This technique provided an accurate and rapid measurement of viability and results were found to correlate closely with the colony-forming unit (CFU) method when used under the same conditions. In addition, the epi-fluorescence method proved to be more versatile in that viability measurements could actually be carried out at low water activities ( a w), whereas the reference CFU method required an increase in a w.

The Relation between Contact Lens Oxygen Transmissibility and Binding of Pseudomonas aeruginosa to the Cornea after Overnight Wear

To assess adverse effects of contact lens-induced hypoxia on the rabbit cornea in vivo and determine the relation between binding of Pseudomonas aeruginosa and oxygen transmissibility for rigid and hydrogel lenses. Six rigid lenses with Dk/Ltotal values between 0 and 97 x 10(-9) (cm/second) (ml O2/ml mmHg) and four hydrogel lenses (Dk/Ltotal 9, 20, 39, 51) were tested. All lenses had 14.0-mm diameters and a thickness (parallel) of 0.12 or 0.15 mm. Tear lactate dehydrogenase activity and tandem scanning confocal microscopy determinations were performed after the lens was worn for 24 hours. Binding of P. aeruginosa then was separately determined by the colony-forming unit method. Scanning electron microscopy was used to confirm in vivo tandem scanning confocal microscopy findings. Lens oxygen transmissibility determines binding of P. aeruginosa to the cornea after the lens is worn for 24 hours; epithelial damage produced by lenses of lower Dk/Ltotal appears to be the dominant biologic factor for P. aeruginosa binding and not lens rigidity. These results suggest that the risk of P. aeruginosa keratitis developing with overnight wear will be enhanced significantly for contact lenses with Dk/Ltotal values less than 50 x 10(-9) (cm/second) (ml O2/ml mmHg) (human equivalent oxygen percentage < or = 15%), and this risk will increase with further decreases in oxygen transmissibility. Because no hydrogel lenses approved by the Food and Drug Administration are available with oxygen transmission at this level, patients should be made aware of the increased risk of infectious keratitis associated with the overnight wear of current extended wear hydrogel lenses. Results of this study also demonstrate that quantitative clinical tandem scanning confocal microscopy imaging and tear lactate dehydrogenase activity measurements can provide prospective, noninvasive methods for assessing the ongoing interaction between contact lens and cornea in vivo.

Degradation of Maize Stored at Reduced Water Activities by Eurotium chevalieri in Relation to Lipid Content

Cultures of a xerotolerant mold, Eurotium chevalieri, were grown on full-fat (FM) and defatted (DM) sterilized maize millings stored at reduced water activities (aw) of 0.80 and 0.74 at 30°C. The mold growth was estimated by colony forming unit (CFU) plate-count method and by ergosterol determination. The growth rates measured by the CFU method showed no difference between FM and DM at reduced aw. However, ergosterol contents were markedly higher in FM which indicated the formation of a higher fungal biomass in media containing lipids. Fungal lipase activity was detected and was shown to be responsible for hydrolysis of lipids to free fatty acids, directly assimilated by E. chevalieri. Under conditions of maize storage at reduced aw, fatty acids released from hydrolysis of maize triglycerides could be the sole available carbon source for fungal growth.

Comparisons and applications of methods for assessing the cellular viability of Blastomyces dermatitidis

Two methods for assessing the cellular viability of the yeast form of Blastomyces dermatitidis were compared to the standard method of enumeration of colony-forming units (CFU) on agar, and shown to have advantages. These methods are fluorescent staining with fluorescein diacetate and ethidium bromide, and conversion of the yeast to mycelial form on agar slide cultures at 25°C. Studies with two strains showed good agreement between the three assays; reproducibility of the two newer methods was also demonstrated. The latter methods are faster, can be used for studying the viability of individual cells (this is not possible with the CFU method), and could be useful in studies testing the effects of host effector cells of antifungal drugs.

Colony-Forming Units and Survival of Irradiated Mice Treated with AET or Endotoxin

When mice are given radiation exposures producing the hematopoietic syndrome, it is assumed that it is the killing of hematopoietic stem cells and the leuko- and thrombocytopenias which ultimately develop that predispose the animals to infection, hemorrhage and death. The colony-forming unit (CFU) has many attributes of a (the) hematopoietic stem cell, and it might be expected that a high correlation should exist between CFU survival and survival of the animal. Some earlier studies have supported this correlation, whereas, others have not. In the present experiments three CFU methods (endogenous, recipient, and donor) have been used to evaluate this correlation in mice in which survival has been increased with AET or bacterial endotoxin. The results show that the different CFU procedures yield somewhat different results, yet under certain conditions the LD50's for AET- or endotoxin-treated mice may be predicted within 2-10% from CFU survival curves. In spite of the good correlation between CFU survival an...