text:
A method is disclosed for isolating a mutant microorganism. The method includes the following steps:
(1) separately microencapsulating individual or small numbers of a microorganism population containing the mutant to obtain a first microdroplet;
(2) thereafter surrounding the first microdroplet, with or without an outer semi-permeable membrane, with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel layer contains an indicator material which reacts to the presence of the mutant microorganism;
(3) culturing the resultant microcapsules so that the microdroplets containing mutant microorganism are able to induce a detectable difference in the indicator material from microdroplets, containing non-mutant microorganisms; and
(4) separating the microdroplets containing mutant microorganisms from those containing non-mutant microorganisms based upon the differences in the indicator material.
1. A method for isolating a mutant microorganism, where the method comprises:
(a) separately microencapsulating individual or a small number of microorganisms from a microorganism population containing the mutant to obtain a first microdroplet;
(b) surrounding the first microdroplet, thereby forming an inner microdroplet, with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating on the microdroplet contains an indicator materials which reacts to the presence of the mutant microorganism;
(c) incubating the gel-coated microdroplets to induce a detectable difference in the indicator material surrounding microdroplets containing mutant microorganisms; and
(d) separating the microdroplets containing mutant microorganisms from those containing non-mutant microorganisms based upon the detectable difference in the indicator material.
2. A method for isolating a mutant microorganism, where the method comprises:
(a) separately microencapsulating an indicator material which reacts to the presence of the mutant microorganism to obtain a first microdroplet;
(b) surrounding the microdroplet with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating contains individual or a small number of microorganisms from a microorganism population containing the mutant;
(c) incubating the gel-coated microdroplets so that the microdroplets containing mutant microorganisms induce a detectable difference in the indicator material; and
(d) separating the microdroplets having mutant microorganisms from those having non-mutant microorganisms based upon the detectable difference in the indicator material.
3. The method of claim 1 wherein the indicator material reacts with a metabolite produced by the mutant microorganism.
4. The method of claim 1 wherein the indicator material reacts with a product formed by a reaction between the mutant microorganism and a substance included in its culture medium.
5. The method of claim 1, wherein the inner microdroplets containing the single microorganisms are formed by:
(a) forming a dilute suspension of said microorganisms in a liquid diluent capable of forming a gel upon subsequent treatment, said dilute suspension having a dilution selected so that there is a high probability that each microdroplet produced from said suspension contains one microorganism;
(b) converting said suspension into gel droplets.
6. The method of claim 5, wherein the outer gel coating is formed by:
(a) forming a dilute suspension of indicator material in a liquid diluent capable of forming a gel upon subsequent treatment;
(b) coating the first microdroplet with the suspension from (a);
(c) ejecting the coated microdroplets into a solution which causes the coating to gel, resulting in a gel-coated microcapsule.
7. The method of claim 1, wherein a semi-permeable membrane is formed around the inner microdroplet by cross-linking surface layers of the inner microdroplet resulting in a microcapsule.
8. The method of claim 7, wherein a semi-permeable membrane is formed around the outer gel-coating by cross-linking surface layers of said outer gel-coating resulting in a double membrane microcapsule.
9. The method of claim 7, wherein the indicator material is a species of cells whose growth is affected by a metabolic product of the mutant microorganism.
10. The method of claim 1 wherein before coating the first microdroplet with an outer gel-coating, the microdroplet is cultured for a sufficient time to enable the individual or small number of microorganisms to multiply and form microcolonies.
11. The method of claim 1 where after coating the first microdroplet with an outer gel-coating, the gel-coated microdroplet is cultured for a sufficient time to enable the individual or small number of microorganisms to multiply and form microcolonies.
12. The method of claim 1, wherein said separation is based on the difference in mass of indicator material between said microdroplets containing mutant and non-mutant microorganisms and comprises equilibrium density centrifugation or sedimentation of a suspension containing said microdroplets.
13. The method of claim 9, wherein said separation is based on the difference in mass of indicator material between said microcapsules containing mutant and non-mutant microorganisms and comprises equilibrium density centrifugation or sedimentation of a suspension containing said microdroplets.
14. The method of claim 1, wherein the step of separating the microdroplets containing mutant from non-mutant microorganisms comprises fluorescent, colorimetric, or radioactive sorting of microdroplets in a suspension containing said microdroplets.
15. The method of claim 1, wherein the separation based on the difference in indicator material between said microdroplets containing mutant and non-mutant microorganisms comprises electrical or magnetic sorting of a suspension containing said microdroplets.
16. The method of claim 1, wherein said microorganisms comprise eucaryotic cells.
17. The method of claim 1, wherein said microorganisms comprise procaryotic cells.
18. The method of claim 1, wherein said microorganisms comprise viruses.
19. The method of claim 1, wherein said microorganisms comprise cell hybrids formed by fusing cells of different species.
20. The method of claim 1, wherein mutant microorganisms are incubated in a medium selected from the group consisting of chemically complex, non-sterile, agricultural, industrial, and other commercial process media.
21. The method of claim 9 wherein said growth is effected by amplifying the detectable difference between the indicator species in microcapsules containing the mutant microorganisms and the non-mutant microorganisms, and the separating step is based on physical characteristics amplified thereby.
22. The method of claim 1, wherein the microencapsulated microorganisms are grown in a laboratory culturing medium.
23. The method of claim 7, wherein the inner microencapsulated microorganisms are grown in a non-sterile, multi-species agricultural or industrial process medium wherein one or more species are found in the native environment.
24. The method of claim 1, wherein the mutant microorganism is a naturally occurring mutant of a wild type microorganism.
25. The method of claim 1, wherein the mutation in the mutant microorganism is artificially induced.
26. The method of claim 25, wherein the artificially-induced mutant is the result of genetic engineering by a biological, biochemical or biophysical process.
27. The method of claim 1 wherein the indicator material is a non-living material.
28. The method of claim 27 wherein the non-living indicator material is selected from the group consisting of labelled or unlabelled antibodies, antigens, enzymes, enzyme substrates and combinations thereof.
29. The method of claim 9, wherein said metabolic product of the mutant microorganism is an essential metabolite for said indicator species, and the microcapsules are incubated under conditions restrictive to growth of said indicator species, whereby the microcapsules containing the mutants have a greater number of indicator cells per microcapsule than microcapsules containing non-mutants.
30. The method of claim 9, wherein said metabolic product of the mutant microorganism is an inhibitor of the growth of said indicator species, and the microcapsules are incubated under conditions permissive for growth of said indicator species, whereby the microcapsules containing the mutants have a less number of indicator cells per microcapsules than microcapsules containing non-mutants.
The present invention relates to a method for isolating mutant microorganisms from a population containing the same by microencapsulation techniques and to coating microcapsules with indicator materials for practicing the methods of this invention.
The enormous size of microbial populations continues to be a great asset in a variety of genetic studies including searches for valuable mutant microorganisms where it is possible to effectively select a rare gene type or mutant microorganism. Mutant cell varieties of a single strain of microorganism (procaryotic, eucaryotic or viral) have classically been isolated by a variety of methods including positive cell "selection" and differential "screening" of individual cell colonies.
Selection is used to isolate mutant varieties of microorganisms when a genetic alteration provides the microorganism with a positive growth advantage over its parental population. For example, acquisition of antibiotic resistance can be used to select such mutants on a nutrient agar surface containing the antibiotic. Another example is the acquisition of a biosynthetic gene enabling the organism to grow in a culture medium that would not otherwise support growth.
Mutants which cause only a small change, a negative change (decrease) or no change in the rate of cell growth are identified by screening of cell colonies. The colonies are produced by multiplication of a single cell. Screening is used to detect mutant microorganism species producing beneficial increases (or decreases) in the synthesis (or the breakdown) of important primary and secondary metabolites for example. However, screening to identify a mutant colony can require examination of tens of thousands of individual colonies to determine the presence of mutants. Replica plating is one screening technique. Thereafter, tedious visual comparison of petri dish pairs are required as well as relatively large amounts of selective and/or restrictive materials which serve to differentiate the mutant from its parent. In general, although screening techniques, are highly effective in achieving the desired result, they are labor and material-intensive requiring examination of many individual colonies, usually in petri dishes.
Recently, a technology has emerged which provides for encapsulating biological material such as living tissue, individual cells, viruses, and biological macromolecules within a semi-permeable membrane. The basic approach in this technique involves suspending the biological material to be encapsulated in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water, e.g., a gel, to provide a temporary environment for the biological material. The medium is formed into droplets containing the tissue and gelled by changing any one of a variety of ambient conditions. These temporary capsules are then subjected to a treatment which results in the production of membranes with a desired permeability (including impermeable membranes). One such technique, is exemplified in U.S. Pat. No. 4,352,883 entitled "Encapsulation of Biological Material", the disclosure of which is incorporated herein by reference.
A description of a technique for separating cells having desired properties from a large population is found in U.S. Pat. No. 4,401,755 entitled "Process for Measuring Microbiologically Active Material" which discloses a method for measuring an unknown quantity of microbiologically active material utilizing unencapsulated microdroplet techniques similar to the first stage process of U.S. Pat. No. 4,352,833. The disclosure of U.S. Pat. No. 4,401,755 is also incorporated herein by reference. After preparing a suspension of gel microdroplets, the suspension is processed in an apparatus having the capability of sensing a physical characteristic of individual gel microdroplets to determine the presence or absence of a desired physical characteristic of the biological material in such a droplet.
It is apparent that a need to develop new mutant isolation techniques exists which will reduce the costs and time spent in selection and screening processes used to isolate mutants from their respective parent populations. Microencapsulation technology, as described in the above referenced patents, provides the potential for solving a variety of problems including the labor and cost excesses of prior art mutant microorganism isolation techniques.
The present invention provides a method for isolating a mutant cell, an otherwise desirable cell, unique cells or cell hybrids (collectively referred to herein as "mutant microorganism") from either its parent population in the laboratory or from a mixed multispecies population such as encountered in agricultural or industrial environments. Single or small numbers of microorganisms from the parent population (containing the mutant which is desired to be isolated) are encapsulated in a semi-permeable membrane by means of microencapsulation techniques. The cells within the microcapsules are cultured, typically dividing and producing substantially pure clones of microorganisms (colonies) within the microcapsules. Either before the growth phase or thereafter, an appropriate diagnostic interactive indicator material, either living or non-living, is microlocalized within a gel layer outside the first microcapsules. The gel layer may optionally, in turn, be encapsulated with an outer semi-permeable membrane to create double microcapsules. Such microcapsules, and the cells within, are then cultured. Using appropriate culturing conditions the microcapsule containing the desired mutant clone will produce a product or products (also termed metabolites) which diffuse outward across the original microcapsule's semi-permeable membrane. When these metabolites encounter the interactive living or non-living indicator material in the external gel coating, physical and/or molecular changes occur which are the basis for detecting and/or isolating the microcapsule containing the mutant microorganism colony. The interactive indicator material may range from unlabelled or labelled molecules, e.g., fluorescent or radioactive antibodies, antigens, enzymes, enzyme substrates, etc. to living cells capable of dividing. In some embodiments simple gel-coated microcapsules are immobilized at the time of assay and the external gel layer converted to a sol. Complexes can thus be assayed in the immediate vicinity of the immobilized microcapsule in a chemically accessible, soluble environment. The microorganism colonies within the microcapsules remain localized and protected for later recovery.
As indicated above, the indicator material is used as the physically or chemically responsive agent for detecting and selecting microcapsules containing mutant microorganisms. A non-inclusive list of characteristics, which can be the basis of identifying and/or separating microencapsulated mutant cells from the microencapsulated parental population, are: increased or decreased indicator cell number, cell mass, cell density or cell size. The amount of an appropriate biochemical indicator substance complexed with a primary or secondary metabolite, diffused outward from a mutant microcapsule, may also be the basis for mutant identification. Based upon such physical and chemical differences among microcapsules, a variety of separation techniques, such as centrifugation, laser-automated microcapsule sorting, microcapsule autoradiography and microcapsule fluorescence screening are available for use.
The present invention overcomes many of the prior art problems associated with isolating mutant microorganisms from parent populations in that, inter alia, it provides in many instances for separation of microcapsules containing the mutants from the microcapsules containing non-mutants without the necessity for classical genetic screening techniques (which involve individual examination of microorganisms) and therefore eliminates the labor and cost excesses of prior art isolation techniques. In other instances, where the parental population also exhibits that characteristic, but to a lesser degree, a particular physical or chemical characteristic, such as the amount of chemical complex formed between indicator material and the diffusible metabolite produced in microcapsules containing mutant cells, is amplified so that the mutant cells within can more easily be separated from a parental population.
The present invention, provides a method for isolating a mutant microorganism from a parent microorganism population containing this same mutant. The method utilizes the relatively new technique of microencapsulation of biological material.
The detection and isolation of mutant cells within microcapsules is described in patent application Ser. No. 580,854, filed Feb. 16, 1984, now U.S. Pat. No. 4,649,109 which is incorporated herein by reference. That application discloses the use of detectable physical and chemical differences (e.g., a change in number of microorganisms per microcapsule) between microcapsules containing the desired mutant microorganisms and those containing non-mutant microorganisms to enrich for and to isolate mutant microorganisms from wild type microorganism populations. This detectable difference serves to enable the discrimination, and/or separation and isolation of microcapsules containing the mutant microorganisms. Several methods for discriminating and isolating microcapsules containing mutant cells are described in the above patent application including sedimentation as well as analytical and diagnostic biochemical methods. The latter include methods in which diagnostic reagents are diffused in and out of the microcapsules to detect a difference in the microcapsules containing mutant microorganisms.
The use of the present invention to obtain a mutant producing a desired product is particularly important to the pharmaceutical industry, petrochemical industry and agriculture in obtaining new and/or improved products.
In the present invention, an indicator material, typically cells of an indicator species of microorganism or, alternatively, indicator molecules, are coated on or microencapsulated around microcapsules containing the species from which the mutant cells derive. One then isolates microcapsules containing mutant microorganisms based upon the diffusion of unique molecules or altered quantities of molecules from the mutant microorganisms interacting with the diagnostic indicator material situated immediately outside the first (or inner) microcapsule. Cumulative differences in the mass of indicator cells, or, in the level of a detectable binding of diffusible metabolite to indicator molecules, are the result of the first microcapsule bearing the mutant cells and thereby provide a basis for identifying and isolating the mutant cells.
The gel-coated or double microcapsular structure allows for the introduction of a species of living cells into the outer gel layer or outer microcapsule for use as an indicator material. The juxtaposition or appropriately chosen cell species allows the detection and isolation of many highly desirable mutant microorganisms from a wild type population. For example, with the introduction of a growth responsive indicator cell species into the outer microcapsule, the presence of mutant cells producing diffusible metabolites can be detected. A mutant may produce increased primary or secondary metabolite levels which cross-feed the indicator cells and thereby stimulate their growth. Alternatively, other mutants may produce increased levels of growth inhibitors which inhibit the division of indicator cells. Still other mutants may increase the proportion of a metabolite being secreted and rendered diffusible and thereby influence the growth rate of appropriately chosen responsive indicator cells. This stimulation or alternatively inhibition of growth of appropriate indicator cells induces a detectable difference (e.g., a change in number of indicator cell microorganism per double microcapsule) between microcapsules containing the desired mutant microorganisms and those containing non-mutant microorganisms. With similarly sized microcapsules, density is highly sensitive to the cell number of a given microorganism population. This detectable difference (such as provided by a difference in indicator cell number) serves to enable the discrimination and/or separation of microcapsules containing mutants from those with non-mutants. Finally, the microcapsules containing mutant microorganisms are isolated from those microcapsules containing non-mutant microorganisms by separation techniques based directly or indirectly on the detectable difference, e.g., a difference in microcapsule density or mass resulting from a change in indicator cell number per microcapsule.
The present invention comprises first, microencapsulating individual microoganisms from a microorganism population which contains the mutant desired to be isolated. Encapsulation of single microorganisms can be accomplished by techniques well-known to those skilled in the art as discussed in detail in U.S. patent application Ser. No. 580,854 now U.S. Pat. No. 4,649,109. In particular, single microorganism encapsulation can be readily achieved by controlling the concentration of the microorganism in suspension so that each microcapsule will receive, on average, one microorganism.
Generally, the microorganism to be encapsulated are prepared in accordance with well-known prior art techniques, as individual (disaggregated) cells are suspended in an aqueous medium suitable for maintaining viability and for supporting the ongoing metabolic processes of the particular microrganism involved. Media suitable for this purpose are available commercially. Similarly, small numbers of microorganisms can be encapsulated in one microcapsule, if so desired.
Microcapsules may be fabricated in virtually any desired diameter with minor diameter fluctuation by appropriate orifice selection. This assures an essentially constant mass among inner microcapsules populated by freely dividing mutant or non-mutant cells.
The microcapsules are formed so that there is a high probability that each microcapsule contains a small number or one unit of microbiologically active material (i.e. microorganism or cell). This can be effected by regulating the dilution of the liquid composition, sol, to be used to produce the microcapsules using knowledge of the size of the microbiologically active material and the predetermined size of the microcapsule to be produced. The regulation of these factors can be determined by conventional Poisson statistical analyses so that the number of microcapsules containing more than the desired number of microbiologically active materials is more than two standard deviations from the mean. It is desirable, for example, to encapsulate zero to one microbiologically active cell per microcapsule in mutant screening and in recombination DNA research (where the object is generally to isolate desirable spontaneous mutant microorganism or genetically engineered microorganisms from a large parental population of such microorganisms).
The preferred encapsulation techinque is that described in the above-referenced U.S. Pat. No. 4,352,883 (Lim). In brief, this approach involves suspending the microorganism to be encapsulated in a physiologically compatible medium containing a water soluble substance that can be made insoluble in water (gelled) to form a temporary protective environment for the microorganisms so encapsulated. The medium is next formed into droplets containing the single microorganism and gelled, for example, by changing ambient conditions such as temperature, pH or the ionic environment. The microdroplets thereby produced may then, preferably, be subjected to a treatment that results in the production of a membrane of a controlled permeability about the shape-retaining temporary capsules.
The microdroplets can be fabricated from any non-toxic, water soluble substance that can be gelled to form a shape-retaining mass by a change of conditions in the medium in which it is placed, and that also comprises plural groups which are readily ionized to form anionic or cationic groups. The presence of such groups in the polymer enables surface layers of the capsule to be cross-linked to produce the desired membrane when exposed to polymers containing multiple functionalities of the opposite charge.
The presently preferred material for forming the microdroplets is a polysaccharide gum, either natural or synthetic, of the type which can be (a) gelled to form a shape-retaining mass by being exposed to a change in conditions such as a pH change or by being exposed to multivalent cations such as Ca++ ; and (b) "cross-linked" or hardened by polymers containing reactive groups such as amine or imine groups which can react with acidic polysaccharide constituents. The presently preferred gum is alkali metal alginate. Other water soluble gums which can be used include guar gum, gum arabic, carrageenan, pectin, tragacanth gum, zanthan gum or acidic fractions thereof. When encapsulating thermally refractory materials, gelatin or agar may be used in place of the gums.
The preferred method of forming the desired semi-permeable membrane about the microdroplets is to "cross-link" surface layers of a gelled gum of the type having free acid groups with polymers containing acid reactive groups such as amine or imine groups. This is typically done in a dilute solution of the selected polymer. Generally, the lower the molecular weight of the polymer, the greater the penetration into the surface of the microdroplet, and the greater the penetration, the less permeable the resulting membrane. Cross-links are produced as a consequence of salt formation between the acid reactive groups of the cross-linking polymer and the acid groups of the polysaccharide gum. Within the limits, semi-permeability can be controlled by selecting the molecular weight of the cross-linking polymer, its concentration, and the duration of reaction. Cross-linking polymers which have been used with success include polyethylenimine and polylysine. Molecular weight can vary, depending on the degree of permeability required, between about 3,000 and 100,000 or more. Good results are obtained using polymers having an average molecular weight on the order of 35,000.
In a preferred embodiment, a semi-permeable membrane is formed around this first microdroplet. This enables single mutant microorganisms to divide within a physical envelope so that whatever physical, chemical and/or biological identity they possess as single mutants (either a constant identity or an inducible property) can be amplified to facilitate the physical separation and recovery of the desired mutant.
Thereafter, these first microcapsules are preferably individually encapsulated in a sol which contains an appropriate diagnostic "indicator material." This indicator material is chosen to interact with primary, secondary, or other metabolites issuing from the mutant microorganisms. The sol consists of the same or similar sol ingredients as contained in the first (inner) microcapsules. The concentration of microcapsules suspended in the sol and the diameter of the sol droplets can be varied to achieve zero to one microcapsule per sol droplet. The sol droplets, which preferably contain zero to one inner microcapsules, are converted to a gel phase by using techniques as described above. The encapsulated first microcapsules may then be incubated under appropriate culturing conditions or alternatively treated with appropriate reagents to form an outer semi-permeable membrane or second microcapsule surrounding the first microcapsules. This results in double membrane microcapsules.
The use of a second, outer semi-permeable membrane has a number of advantages. The sterile environment can be maintained inside the capsule even when there is a non-sterile culture environment outside. Another advantage to using an outer membrane is that the gel may be converted to a sol thereby facilitating chemical diffusion, mixing, cell growth, and assay of molecular complexes in the sol phase. The presence of an outer membrane eliminates the need to physically immobilize individual microcapsules prior to converting the external gel coating to a sol phase for biochemical or immunological assay experiments. The concentric microcapsule structures may be passed through a liquid phase automated cell sorting device to detect fluorescent or otherwise labeled biochemical complexes, etc. within the microcapsules. Alternatively, these labeled microcapsules may be displayed on a two dimensional surface for fluorescence detection, autoradiography, or for other assays.
Choice of the diameters, permeabilities, and chemical composition of the two respective (inner and outer) double microcapsule membrane will depend upon the particular experiment being carried out. The choice of culture media must first permit the growth of cell species from which the mutant is derived. Either simultaneously or subsequently the medium plus the metabolite synthesized and diffused outward by the desired mutant must support growth of the indicator cells. Alternatively, if the mutant is producing a growth inhibitor, such as an antibiotic which reduces the rate of growth of appropriate indicator cells, then the culture medium without this antibiotic should support normal growth of the indicator cells. The growth phase of the indicator cells may be planned to coincide or alternatively follow the growth of the microorganism cell species from which the mutant cells are derived. It is also within the scope of the present invention to include applications in which the microcapsular placement of the mutant cells and the indicator material is reversed. That is, the indicator material could be placed in the inner microcapsule while the cell species containing the mutant is placed in the outer microcapsule.
Isolation of many classes of mutant microorganisms can be achieved by the present double encapsulation process, including spontaneous or induced mutants, cell hybrids formed by uniting cells of different species, and genetically engineered species carrying exogenously introduced genes. The isolation procedure takes advantage of the changes in metabolites and/or their concentrations produced by, and emerging from these mutants. The mutants to be selected synthesize and secrete, or outwardly diffuse, novel, changed, increased or decreased amounts of primary or secondary metabolites. Single or small numbers of cells of one species from which the mutant(s) is derived are cultured to form microcolonies within the microcapsules. These mutants synthesize a wide variety of different metabolites. Although it would be difficult to isolate such a mutant by itself, by choosing the appropriate indicator material, and then selecting for the microcapsule's physical or chemical alteration brought about by that indicator as disclosed herein, the mutant can readily be isolated.
An indicator is chosen that reacts to the presence of the metabolite. In a preferred embodiment, cell species in which the growth rate of the cell is affected by the metabolite are used as indicator material. The choice of a particular indicator species will be determined by the type of mutant one is seeking to isolate, and based upon that goal, the particular species can readily be determined by the skilled practitioner. For example, by choosing as an indicator a cell that has an increase in its rate of growth as a result of exposure to a novel or an increased level of an amino acid, microcapsules containing a mutant producing or overproducing an amino acid can be readily identified. The auxotrophic, amino acid requiring indicator cell species is microencapsulated around the original microcapsules carrying the first cell species. An appropriate culturing period is allowed for growth in a medium which is deficient in the particular amino acid produced by the mutant but otherwise compatible with growth of the indicator cell species. The inner microcapsule membrane is formed so as to permit secretion (or leakage) of the amino acid metabolite from the overproducer mutant cell species. The culture period should allow for a number of generations of indicator cell growth so that the number of indicator cells around mutant microcolonies will be sufficiently different from the number around non-mutant microcolonies to enable detection and/or separation of the mutant microcolonies. The various detection and separation methods are techniques well-known in the art.
For instance, the difference in indicator cell number between microcapsules containing mutant and non-mutant cells permits both simultaneous (bulk or in toto) separation techniques as well as sequential (or serial) separation techniques. Examples of bulk separation include equilibrium density centrifugation, velocity or gravity sedimentation, separation in electrical or magnetic fields, chromatography, etc. Examples of serial techniques include detection of individual microcapsules via radioactive, luminescent, fluorescent or colorimetric labels, etc. Serial detection can be accelerated utilizing automated devices such as laser and automated particle sorting devices.
This present invention can also be used to isolate industrial strains of microorganisms that have been mutated or "improved" to synthesize greater yields of secondary metabolites such as antibiotics. Microencapsulation of single or small numbers of such cells and subsequent re-encapsulation with indicator cells sensitive to the metabolite, e.g., antibiotic, provide a useful system for selecting these improved mutants. The growth rate of the indicator cells reflects the relative amount of antibiotic secreted by the cells under investigation. A selection based on the number of indicator cells in a microcapsule following a growth phase can be performed using mass separation methods, e.g., equilibrium or velocity sedimentation. Alternatively, indicator cell yield per microcapsule may be determined by techniques such as laser automated particle sorting methods which are well known to those familiar with this field.
In another application of this invention, mutants may be selected which synthesize and export novel or increased levels of macromolecules, such as proteins, carbohydrates, lipids, and mixed complexes thereof. Protein macromolecules may include enzymes, peptides, hormones, other protein "factors", serum proteins, etc. These mutants are isolated by using indicator cells that exhibit a changed growth rate in response to the exported macromolecule in a manner similar to those examples discussed above. Alternatively, increased production levels of macromolecules may be detected by using indicator materials, rather than living cells, placed in a gel layer around the original microcapsule containing the mutant cells. An indicator material could be an antibody directed against and specifically binding to a desired macromolecule.
This general technique also enables one to select mutants which fail to produce or produce lower amounts of a specific, undesirable or detrimental metabolite. Similarly, mutants producing macromolecules having increased or decreased biological or biochemical specific activities may be produced.
Selection of mutants producing, or overproducing levels of a desired active protein factor, such as an enzyme utilizing a modification of the above basic method, is straightforward. The desired enzyme will convert one or more appropriate precursor substances (described herein as "A") to a product (termed "B"). An indicator cell species is placed in the gel layer surrounding the original microcapsules containing the parent cell population from which the mutant is derived. The indicator species is chosen to respond with a change in growth rate to the increased concentration of B produced by the mutant cells. Substance A, the substrate for the enzyme, may or may not be diffusible through the microcapsule membranes. If it is not diffusible, then it will be co-encapsulated at sufficient concentration, together with the original cells (including the mutant) so that the resultant concentration of product B will be sufficient to cross-feed or alternatively inhibit the indicator cells. If substance A is diffusible, it may be simply added to the microcapsule culture medium. Substance A is converted to product B by the target enzyme. Product B must be diffusible from the inner microcapsule so it can interact with the indicator material. Selection of mutants is based upon detecting the "change" in the indicator material, e.g., a cell mass increase, as a result of its "reaction" with product B. The indirect method of using an external substate source for revealing elevated enzymes levels in mutant cells contrasts with the direct process described earlier in which their mutant cells are selected by the increased synthesis of primary or secondary metabolites (which directly affect indicator cells). This method can be used to select for mutants producing large amounts of a desired macromolecule.
Target enzymes having commercial importance include the hydrolases which are principally derived from the genera Asperqillus and Bacillus. The extra-cellular secreted hydrolases overproduced by these mutant cells will generally cause increased indicator cell growth rate as a result of cross-feeding of indicator cells with hydrolysis products of proteins (amino acids), polysaccharides (mono- and disaccharide products) and fats (small fatty acids). These hydrolysis products (B) are microcapsule-diffusible. The alteration of indicator cell growth rate and thereby indicator cell mass, permits the detection and separation of microcapsules containing the mutant cells as described above.
In a similar manner, mutant microorganisms may be selected which produce particular enzymes having increased specific activities, increased thermal stabilities, altered substrate specificities or alterations in other intrinsic properties. The microcapsule selection process merely requires that an appropriate indicator material (e.g., living cells) has an opportunity to react, i.e., grow, with product B under culturing conditions which will differentiate between the microcapsules producing the mutant enzyme and microcapsules producing normal enzyme.
This method can be used to isolate a wide variety of mutant microorganisms as shown in the following table. The mutant synthesizes an enzyme which converts substance A to product B. Product B cross-feeds indicator cells which are chosen because they require that nutrient for growth. Alternatively, product B is an inhibitor of cell growth so that increased conversion of substance A to B by mutant microorganism will decrease the indicator cell growth allowing isolation of microcapsules containing mutant microorganisms.
TABLE__________________________________________________________________________Enzyme A B Indicator Cells__________________________________________________________________________(1) L-amino acid X Acylated D, L-amino acid X amino acid X aminoacylase L amino acids auxotroph (racemic mixture)(2) aspartase ammonium fumarate L-aspartic acid aspartate auxotroph(3) Penicillin penicillin G 6-aminopenicil- pencillin acylase lanic acid sensitive type I(4) Pencillin pencillin V 6-aminopenicil- pencillin acylase lanic acid sensitive type II(5) Cephalosporin 7-aminocephalo- cephalothin cephalosporin acylase sporanic acid sensitive (7-ACA)(6) Cephalosporin (7-ADCA) cephalexin cephalosporin acylase sensitive(7) Tryptophan indole, pyruvate tryptophan tryptophan synthetase and ammonia auxotroph(8) Tryptophan indole and serine tryptophan tryptophan synthetase auxotroph(9) --tyrosinase phenol, pyruvate L-tryosine tyrosine ammonia auxotroph(10) steroid steroid steriod, e.g., ergosterol transformation precursor(s) ergosterol requiring biosynthetic yeast mutant enzymes(11) vitamin vitamin vitamins vitamin- biosynthetic precursors requiring enzymes microorganism__________________________________________________________________________
Microorganisms containing exogenous DNA, e.g., vectorially introduced cloned DNA sequences, can also be readily screened. E. coli cells transformed with a mammalian gene for human growth hormone (hGH) are microencapsulated within semi-permeable membranes having porosity sufficient to let the hGH protein through. The microcapsules are reencapsulated in a gel containing mammalian target cells which require the growth hormone for their own growth. The microcapsules are cultured in mammalian growth medium containing a bacteriostatic antibiotic which prevents any free E. coli cells from growing and contaminating the medium. After an appropriate culturing interval, hormone-secreting E. coli cells containing the cloned DNA sequence should contain a greater number of mammalian cells than microcapsules containing E. coli which do not secrete the hGH. The desired microcapsules can be isolated by automated cell-sorting techniques which discriminate microcapsules containing large numbers of mammalian cells from those with only a few mammalian cells. Alternatively, equilibrium or velocity sedimentation can be used to fractionate the microcapsules, based on density difference between microcapsules carrying many versus few cells by techniques well-known to those skilled in the art.
Bacterial mutant strains which accumulate increased amounts of soluble inorganic nutrients (such as phosphate) and convert them to insoluble products (such as polyphosphate) can be easily screened. Such mutants of Acinetobacter and other species are detected by using the reversible nature of this phosphate conversion process coupled with phosphate diffusion cross-feeding. Under aerobic conditions, the cells accumulate insoluble polyphosphate whereas anaerobic cultures hydrolyze this polyphosphate to produce soluble inorganic phosphate. Microcolonies of these cells are first cultured in microcapsules in an aerobic phosphate-rich medium. Subsequently, the microcapsules are re-encapsulated. The outer microcapsules contain a number of low phosphate or phosphate starved cells of any appropriate indicator species (all species require phosphate to grow). The double encapsulated cells are then cultured in an anaerobic, phosphate-free, or very low phosphate, medium which would support growth of the indicator cells, but for the lack of phosphate. Microcapsules containing large amounts of polyphosphate which can be converted to soluble inorganic phosphate to support growth will have greater indicator cell growth. The microcapsules are then assayed and isolated based on indicator cell number. Those possessing the largest number of indicator cells should contain Acinetobacter cells overproducing polyphosphate. This is particularly valuable in obtaining strains that are helpful in agriculture.
Cells secreting new, or increased, levels of catabolic enzymes capable of metabolizing industrial intermediates, synthetics or waste products can also be isolated. The solubility of industrial products such as petrochemicals in aqueous solvent is limited in a number of instances and often the density of these substances is not identical to that of aqueous growth medium. By placing an appropriate amount of such an industrial product in the outer microcapsule and including one or a small number of potential mutant cells within the inner capsule, the metabolism of the product can be followed as the cells grow. The close proximity of the cells to the industrial product favors its metabolism by catabolic enzymes secreted by cells within the same double microcapsule. A poor external growth medium may be desirable to facilitate detection of growth which is enhanced by metabolism of the microencapsulated industrial product. The microcapsules containing mutants metabolizing the industrial product show both an increased cell number and a reduced amount of the original product. These changes e.g., density changes or increased fluorescent cell labelling, are then used to detect and isolate the mutants.
With a similar objective, previously undiscovered or difficult to obtain microorganisms capable of metabolizing, degrading or detoxifying natural or man-made substances, such as organic waste chemicals, petrochemicals, and agricultural by-products, can be isolated from a mixed population of microorganisms. The chemical breakdown of one of these products (termed substance X) by such a microorganism may or may not affect the growth rate of that microorganism. If growth rate is affected, then the microorganism may be isolated by the microencapsulation of single cells followed by growth in the presence of substance X, as described in U.S. patent application Ser. No. 580,854 now U.S. Pat. No. 4,654,109. If not, the microorganism of interest is isolated based upon its ability to metabolize substance X and the use of the present invention. A metabolic break-down product of substance X provided by the microorganism is used as a nutrient for the growth of another species of microorganism. This second species of microorganism is used as the indicator microorganism. Cells of the original undefined population are grown in appropriate culture medium within microcapsules to form microcolonies. These microcapsules are then re-encapsulated to form double microcapsules each containing indicator microorganisms. These microcapsules are cultured in medium lacking an essential nutrient for growth of the indicator cells. The nutrient will be furnished by the metabolic breakdown of substance X. Indicator cells will selectively grow in those microcapsules which contain an internal microcapsule colony species which is metabolizing substance X (substance X must either be diffusible or be encapsulated originally with the single cells). After a sufficient period of time for growth of the indicator cells, the microcapsules containing larger numbers of indicator cells are separated and the mutant species isolated.
Metabolic interactions between various pairs of microorganisms can also be studied using the present process. By controlling the semi-permeable membrane porosity, an investigator can study cross-feeding of different molecular weight metabolites between the different species.
In the process of forming gel-coated microcapsules, depending upon the volume of sol coating component relative to the inner microcapsule component and upon the final droplet volume, a certain fraction of the gel droplets formed will not include an original microcapsule. These "empty" droplets will not affect mutant microorganism selection because the selection depends upon the interaction of mutant microorganisms with the indicator material. The sol to gel conversion step involving the microcapsule coating is the same process used to form the original microcapsule gel droplets. The present invention includes use of double layered gel microdroplets in mutant isolation where the inner, the outer or both potential semi-permeable membranes surrounding the respective gel layers are absent. Therefore, in one form of the present invention, two gel layers without membranes are present. One layer contains the microorganism species providing mutant cells and the other layer contains the indicator material. This material may include chemical substances, biochemicals, as well as species of living cells different from the mutant as described above. The embodiment where there is no inner semi-permeable membrane is referred to as a gel-coated microdroplet or microencapsulated microdroplet.
When coating the inner microdroplet, care must be used so that the inner microdroplet is not reliquified. This can be done by techniques well known in the art. For example, one can use two different types of gelling, e.g., a calcium gel for the inner microdroplet and a low-melting temperature thermal gel for the outer coating.
However, the use of semi-permeable membranes to regulate diffusion in and out of gel microdroplets and to provide physical barriers for microorganism containment, i.e., sterility, is sometimes essential. These semi-permeable membranes often facilitate microorganism mutant isolation as described above by excluding undesirable substances (e.g., destructive proteases or other cells), by permitting selective passage of desired substances (e.g., diffusion of a metabolite from the mutant cells to indicator cells) and by retaining and thus concentrating desired substances (e.g., use of the outer microcapsule membrane to retain the metabolite from the mutant cells to enhance its effect on indicator substances).
In one embodiment, the outer gel coating is treated with reagents to form an outer semi-permeable membrane as described for the inner microcapsules. The semi-permeable outer membrane, whose porosity and composition may be controlled and may be different from the inner microcapsule membrane, can be used for a number of functions. These functions include: (I) maintaining sterility of the microcapsule in non-sterile culture environments; (II) allowing diffusion of culture medium nutrients to the indicator cells; (III) restricting loss to the culture medium by diffusion of the targeted metabolite produced by the mutant microorganisms (i.e., the metabolite of interest must be diffusible between the inner microcapsule and the outer microcapsule so that it can reach the indicator microorganisms outside the inner microcapsule membrane); and (IV) providing a physical constraint which permits conversion of the gel phase back to the sol phase (for reasons of cell growth, biochemical assays, etc.) without the consequent loss of indicator cells to the surrounding culture medium.
The diameter and thus the volume of the gel coating applied to the microcapsules can be controlled by the ejection orifice diameter and sol viscosity. There is no difficulty with coating the inner microcapsule with an outer gel layer. The microcapsules are sturdy and can readily be ejected after coating. The ratio of outer gel volume to inner microcapsule volume is chosen to suit the experimental application. Thus if the objective of the gel coating is to immobilize macromolecules outside the microcapsule then a thin gel coating will often suffice. If the objective is to allow for cross-feeding and subsequent growth of an indicator species of microorganism within this gel volume then a much thicker coating may be required. If growth of the indicator microorganism (in response to metabolites produced by a mutant colony inside) is to be used for direct physical separation of the mutant-containing microcapsules (e.g., by density sedimentation) then the second capsule must be large enough so that the mass of indicator cells grown in the outer gel layer will alter microcapsule density to allow such a separation as described above. For such a microcapsule density shift to occur, the ratio of gel coating volume to microcapsule volume should range between 0.5:1.0 and 100:1.0. Within this range, the specific ratio is determined by the growth yield (mass) of indicator cells per volume of gel coating (growth density). The growth yield is determined by nutritional factors. In the cross-feeding situations discussed herein, the production level of a desired growth stimulating metabolite from the mutant cells limits this growth yield.
Automated laser sorting may be employed to separate microcapsules containing varying numbers of indicator cells. With chromatic or fluorescent staining of indicator cells the selection method can be sensitive to small overall numbers of indicator cells per microcapsule wherein the indicator cell numbers vary among the microcapsules. That is, in experimental situations which may not generate sufficient indicator cell growth mass to allow density separation of the microcapsules containing the mutant cells, the automated laser sorting method provides a possible alternative separation method. The difference in cell number of the indicator species among the microcapsules still remains the basis for identifying microcapsules containing mutants in the laser method.
The present invention also provides a kit for practicing the methods described above. The kit comprises the ingredients or components required to form microcapsules on a laboratory or research scale to screen for mutants in accord with the present invention. As such, the kit comprises a container or package having quality-controlled reagents therein: sterile alginate solution, sterile 2-(cyclohexylamino)ethane sulfonic acid, indicator material, sterile solution of polylysine having predetermined molecular weight and a sterile solution of polyethylenimine (PEI) having a predetermined molecular weight. The molecular weights of the polylysine and PEI are predetermined to produce microcapsules having a desired permeability in accord with the known technology as described in, for instance, U.S. Pat. No. 4,352,883. In addition, sterile CaCl2 and mechanical devices for forming microdroplets can be supplied as part of the kit. Preferably, the solutions are provided in sealed sterile vials having sufficient quantities for one experiment. Further, it is preferred that the solutions comprise physiological saline and polylysine and PEI solutions also contain 0.2M MOPS.
From the foregoing, it is apparent that isolation of mutant microorganisms in accordance with the present invention, can be practiced on a wide variety of species, using a variety of techniques that result in a difference between indicator material in microcapsules containing mutants and those microcapsules that do not.
It is appreciated that those skilled in the art upon consideration of this disclosure, may make modifications and improvements without departing from the spirit and scope of this invention.
Patent Citations
Referenced by
Legal Events
Original Image


text:
2 posts tagged with parody by ciderwoman.
2 posts tagged with parody by ciderwoman.
Displaying 1 through 2 of 2.
bohemian election
Bohemian election. Just a little something to lighten the day. Bound to be a double post but can't find any signs, so apologies in advance if it is. Altogether now, My brother jeb has votes put aside for meeee...
It's Rodeohead,
It's Rodeohead, (MP3 download), the radiohead country and western medley. Please note there is absolutely no reason to post this apart from it's Friday, it's a bank holiday weekend and it made me laugh. If you're looking for in-depth then move along, nothing to see here...
Page: 1

subset: commoncrawl


text:
School Superintendent Praises Police Command StaffThursday, October 16, 2014
Police Commission Chairman Paul Mangiafico has told Police Commission members that the superintendent of schools has commended Newtown Police Department command staff members for their work following the October 1 bomb threat received at Sandy Hook Elementary School in Monroe.
Phase 4: School Board Approves SHS Construction DocumentsFriday, October 10, 2014
The Board of Education approved on Tuesday, October 7, the fourth phase of the Sandy Hook School building construction project to be forwarded to the state.
Previously, at its September 16 meeting, the school board heard a presentation from Diversified Project Management's Geralyn Hoerauf, Consigli Construction's Aaron Krueger, and Svigals + Partners' Julie McFadden, who each reported on the progress of the Sandy Hook School building project. Phase 4 of the project will focus on building construction.
Five Agencies Probing 'Terroristic' Bomb Threat Against SHESThursday, October 2, 2014
MONROE — At least five law enforcement agencies are investigating a telephoned bomb threat to Sandy Hook Elementary School at 375 Fan Hill Road, which was received on the morning of Wednesday, October 1.
The incident amounts to "a terroristic act" heavily punishable by federal criminal law, according to a spokesman for Monroe Police Department.
Five Agencies Probing 'Terroristic' Bomb Threat Against SHESThursday, October 2, 2014
MONROE — At least five law enforcement agencies are investigating a telephoned bomb threat to Sandy Hook Elementary School at 375 Fan Hill Road, which was received on the morning of Wednesday, October 1.
The incident amounts to "a terroristic act" heavily punishable by federal criminal law, according to a spokesman for Monroe Police Department.
First Selectman: SHES 'Students And Staff Are Safe'Wednesday, October 1, 2014
Newtown First Selectman Pat Llodra has issued a statement in response to the bomb scare called in to Sandy Hook Elementary School this morning:
SHS Students Find Astronaut Acquaintance Is A Down-To-Earth GuySaturday, September 20, 2014
During the 2013–14 school year, Sandy Hook School third and fourth grade students had the opportunity to talk, via Skype, with two flight engineers aboard the International Space Station (ISS), and on Friday, September 10, one of the astronauts visited the school, offering presentations and visits with students during the morning.
Design Elements Presented: School Board Updated On SHS ProgressFriday, September 19, 2014
Blues, greens, tans, and grays made up the primary colors of the proposed interior and exterior materials for use in the new Sandy Hook Elementary School that were presented to the Board of Education at its meeting September 16. The update came after the materials had already been presented to the Public Building & Site Commission.
A Special School Preview For KindergartenersThursday, August 28, 2014
Kindergarten students across the district had a taste of the school year on Friday, August, 22, when they experienced riding on a school bus for the first time in their public school careers.
At Hawley Elementary School, students were greeted by their teachers, parent volunteers, and Principal Christopher Moretti. The children visited their classrooms, where they could color and make themselves familiar with the spaces.
P&Z Approves Sandy Hook School ProjectThursday, August 7, 2014
Following an hourlong public hearing on July 31, Planning and Zoning Commission (P&Z) members unanimously approved redevelopment plans for the proposed new Sandy Hook Elementary School at 12 Dickinson Drive, off Riverside Road.
The new school would replace the former Sandy Hook School, which the town demolished last year following the December 2012 massacre there.
The P&Z's zoning approval marks a major local hurdle for the construction project.
Local Residents Join Anti-Christie Protest In GreenwichFriday, July 25, 2014
Dave Stowe, vice chairman of Newtown Action Alliance, joined more than 50 Newtown, Sandy Hook, and area residents in Greenwich on Monday, July 21, where at least an additional 150 people gathered to protest the appearance of New Jersey Governor Chris Christie. Gov Christie was fundraising in the Bell Haven area of Greenwich for Connecticut Republican gubernatorial candidate Tom Foley.


text:
Generic WLAN Connection Instructions
ITS provides support for connection to the CWU WLAN only for those platforms listed on the wireless web site. However, it may be possible for you to connect your unsupported device to the network utilizing the information in this document. Since these instructions are generic, some technical knowledge of your operating system is required, and ITS offers no additional support nor any guarantee that your device will function on the WLAN.
CWU uses two levels of security on the WLAN − device registration and EAP authentication, so two steps are necessary to get connected:
The least error-prone method of registration is via the wireless network itself. Registration services are available on an open network with SSID 'hostreg'. Connect to hostreg and access http://hostreg.cwu.edu. Enter your typeless distinguished network (eDirectory) username (e.g. .joliea.j.students.cwu) and your password and click the login button. The system should automatically detect the address of your wireless interface and display it. It will also attempt to determine your operating system based on your browser. You will need to select the building where you will most often use the device and the wireless category from the drop-down lists. If the device is CWU property, enter its tag number. Then click the Agree & submit button.
If your device does not have a browser or you prefer not to register wirelessly, you may access the registration system from a lab or office computer. If you do so, you will need to manually enter the address for your wireless interface. How you retrieve that address is platform specific. You will also need to manually select the operating system of the device. Otherwise the registration process is the same as above.
Once you have registered your device, you will need to wait until the next quarter hour before it will function on the network. A process runs every 15 minutes and updates the servers which provide DHCP services for the university.
CWU uses EAP (Extensible Authentication Protocol) for authentication and dynamic generation and rotation of WEP keys. The IEEE 802.1x standard defines a framework for various implementations of EAP. The EAP method used by CWU is EAP-TTLS/PAP. Other EAP methods will not work in our environment. You will need a piece of software known as a supplicant which supports EAP-TTLS/PAP. This software is integrated in some operating systems and/or driver sets, is available in Open Source distributions for others, and is found in several commercial products. Install the supplicant and configure it as follows:
The SSID you will need to connect is 'cwu'. This SSID is not broadcast, so you will not be able to browse for it.
Most EAP-TTLS supplicants support multiple tunneled authentication methods. Be sure you select PAP. Other methods will not work.
EAP-TTLS permits the specification of an outer username. Set this to 'anonymous'.
For the inner credentials which are actually used for authentication, use your network (eDirectory) username and password. Use the short form of your username without the context (e.g. joliea).
Do not enter a WEP key. Configure the software to use the WEP key automatically generated during the EAP authentication process.
In the CWU environment EAP-TTLS uses certificates on the server side only. You do not need to generate a client-side certificate, but you will need to configure your system to accept and verify certificates from the authentication servers. The two RADIUS servers are asherah.cts.cwu.edu and anat.cts.cwu.edu. Their certificates are self-signed, so you may need to add them to your certificate store for them to be accepted. It is important to verify certificates for the authentication servers in order to prevent connection to a rogue access point.


text:
Recent answers to Why does Gmail look so bad in Firefox 18? It looks like bad anti-aliased fontshttps://support.mozilla.org/en-US/questions/9471862013-03-20T11:49:08-07:00I found this and it worked for me: 1. type about:config in address bar 2. search for "gfx.content.az2013-03-20T11:49:08-07:00JoeSalazarhttps://support.mozilla.org/en-US/questions/947186#answer-418970
I found this and it worked for me: 1. type about:config in address bar 2. search for "gfx.content.azure.enabled" and set it to false 3. restart your browser
One other thought on this. My Canon printer died and I bought a new Epson and installed the Epson p2013-03-07T19:45:26-08:00ricktayhttps://support.mozilla.org/en-US/questions/947186#answer-415116
One other thought on this. My Canon printer died and I bought a new Epson and installed the Epson printer the same week I started noticing the blurred fonts in Firefox v19.0.2.

Is there any chance the Epson printer installed any type of fonts or font rendering software that may have caused this problem?
I have Firefox 19.0.2 installed and this problem exists on Google News which I use as a home page. 2013-03-07T19:41:38-08:00ricktayhttps://support.mozilla.org/en-US/questions/947186#answer-415115
I have Firefox 19.0.2 installed and this problem exists on Google News which I use as a home page. I never noticed it until I installed this new version a few days ago. At first the fonts look good, but as I scroll down the page the fonts start blurring. Oddly, I found that if I select/highlight the text, the reverse color effect fixes the fonts and when I deselect the text if looks fine again.

Until I start scrolling through the page, and once again the text starts blurring. I once had a prescription for klonopin that caused a similar effect while working at my computer, but I am not tripping on anything when this text blurs. I have never seen this blurred text problem on anything but a google site.

In short, this problem is not fixed in v19.0.2 and in fact I never had the problem until I upgraded. I have used Firefox as my default browser for years but the last three or four versions have tried my patience with stupid design decisions and now this may make me leave it all together.

Anyone know any new fixes for this problem?
Good news, I tried the Firefox 19 release on the Beta Channel and the problem is fixed ! 2013-01-29T14:00:23-08:00fleuryantoinehttps://support.mozilla.org/en-US/questions/947186#answer-403007
Good news, I tried the Firefox 19 release on the Beta Channel and the problem is fixed !
Damned, I have spent the whole day thinking it was due to a hardware problem on my MacBook Pro ( bec2013-01-29T12:32:48-08:00fleuryantoinehttps://support.mozilla.org/en-US/questions/947186#answer-402990
Damned, I have spent the whole day thinking it was due to a hardware problem on my MacBook Pro ( because I replaced the logicboard yesterday), but it apparently wasn't !

I have an awfull aliasing on the google products ( gmail / youtube...) and some other websites but not all. It works well with video sites like dailymotion / vimeo...

What I found is: When I reflesh a youtube page without seeing the video windows, the fonts become clean, if I open a new video page or if I do the refresh at the bottom of the page where we can see the video, the fonts become ugly...

I tried to clean the cache / cleaning my font library but nothing solve the problem..

I tried the old Firefox 17 and the problem disapears...

My computer is an old MacBook Pro from 2007 (A1211 2,33Ghz with 256Mo GPU, 3Go ram) with SnowLeopard and Firefox 18.

I hope they will solve the problem in the next release...

See attached a test with Firefox / Safari / Chrome on mac...(It's easier to see the difference on the imagesharck hosted file bellow, but in reality than worst)

http://img59.imageshack.us/img59/7404/capturedecran20130129a1.jpg
Something is definitely broken in FF 18. Fonts are blurry. I wonder why so few people complain about2013-01-22T03:13:21-08:00Avi11https://support.mozilla.org/en-US/questions/947186#answer-400448
Something is definitely broken in FF 18. Fonts are blurry. I wonder why so few people complain about this. Seems like most people don't care. I can replicate this on any of my 6 Windows machines. For now the only solution is to revert back to FF 17. If it won't be fixed, I'll have to abandon my favorite browser and switch to Chrome, and I do not want this.
Nope didn't work but I really appreciate your determined efforts to help :) 2013-01-19T09:14:36-08:00AshwinJayhttps://support.mozilla.org/en-US/questions/947186#answer-399546
Nope didn't work but I really appreciate your determined efforts to help :)
Try to reset the page zoom on pages that cause problems. View > Zoom > Reset (Ctrl+0 (zero); 2013-01-17T19:21:44-08:00cor-elhttps://support.mozilla.org/en-US/questions/947186#answer-398991
Try to reset the page zoom on pages that cause problems.

View > Zoom > Reset (Ctrl+0 (zero); Cmd+0 on Mac)

http://kb.mozillazine.org/Zoom_text_of_web_pages

You can remove all stored data from a specific domain via "Forget About This Site" in the right-click context menu of an history entry (Show All History or History sidebar) or via the about:permissions page.

Using "Forget About This Site" will remove everything like bookmarks, cookies, passwords, cache, history, and exceptions from that domain, so be cautious and if you have a password or other data from that domain that you do not want to lose then make a note of those passwords and bookmarks.
 You can't recover from that "forget" unless you have a backup of the affected files.

It doesn't have any lasting effect, so if you revisit such a 'forgotten' website then data from that website will be saved once again.
Thanks, but the tuner didn't seem to make any diff. 2013-01-17T13:26:24-08:00AshwinJayhttps://support.mozilla.org/en-US/questions/947186#answer-398905
Thanks, but the tuner didn't seem to make any diff.
Try: Anti-Aliasing Tuner: https://addons.mozilla.org/firefox/addon/anti-aliasing-tuner/ Does it he2013-01-16T22:45:18-08:00cor-elhttps://support.mozilla.org/en-US/questions/947186#answer-398709
Try:

Anti-Aliasing Tuner: https://addons.mozilla.org/firefox/addon/anti-aliasing-tuner/

Does it help if you select another font to display the text?

You can use this extension to see which fonts are used for text that is selected (right-click context menu: Show fonts in selection).

fontinfo: https://addons.mozilla.org/firefox/addon/fontinfo/

You can do a font test to see if you can identify corrupted font(s).

http://browserspy.dk/fonts-flash.php?detail=1

You can try different default fonts and temporarily disable website fonts to test the selected default font.

Tools > Options > Content : Fonts & Colors > Advanced

[ ] "Allow pages to choose their own fonts, instead of my selections above"

No diff after that. 2013-01-16T22:34:36-08:00AshwinJayhttps://support.mozilla.org/en-US/questions/947186#answer-398704
No diff after that.
Does disabling hardware acceleration and closing and restarting Firefox has any effect? 2013-01-16T20:39:14-08:00cor-elhttps://support.mozilla.org/en-US/questions/947186#answer-398658
Does disabling hardware acceleration and closing and restarting Firefox has any effect?
Screenshots of my Gmail and another website attached for comparison. Notice that Gmail text looks bl2013-01-16T10:42:21-08:00AshwinJayhttps://support.mozilla.org/en-US/questions/947186#answer-398506
Screenshots of my Gmail and another website attached for comparison.

Notice that Gmail text looks blurred - as if Cleartype is not working well?

Both were taken while viewing the sites using FF 18.

Just checked. hardware acceleration is on.
Can you attach a screenshot? http://en.wikipedia.org/wiki/Screenshot https://support.mozilla.org/kb2013-01-15T20:58:17-08:00cor-elhttps://support.mozilla.org/en-US/questions/947186#answer-398294
Can you attach a screenshot?

http://en.wikipedia.org/wiki/Screenshot

https://support.mozilla.org/kb/how-do-i-create-screenshot-my-problem

Use a compressed image type like PNG or JPG to save the screenshot.

Try to disable hardware acceleration in Firefox.

Tools > Options > Advanced > General > Browsing: "Use hardware acceleration when available"

https://support.mozilla.org/kb/Troubleshooting+extensions+and+themes

https://hacks.mozilla.org/2010/09/hardware-acceleration/

Try to set the gfx.content.azure.enabled pref to false or if this didn't help disable Direct2D by setting the gfx.direct2d.disabled pref to true on the about:config page and leave hardware acceleration otherwise enabled.

http://kb.mozillazine.org/about:config

It was false. I set it to true and restarted. Still the same. It's only Gmail and Google Drive that 2013-01-15T14:34:26-08:00AshwinJayhttps://support.mozilla.org/en-US/questions/947186#answer-398144
It was false. I set it to true and restarted. Still the same. It's only Gmail and Google Drive that looks weird.
Do you have smoothing turned on as well? Have you reset Firefox? Refresh Firefox - reset add-ons and2013-01-15T13:48:38-08:00Tylerdownerhttps://support.mozilla.org/en-US/questions/947186#answer-398132
Do you have smoothing turned on as well?

Have you reset Firefox? Refresh Firefox - reset add-ons and settings


text:
OLCC's Roseburg office will be CLOSED Monday & Tuesday, March 30-31, as they are moving to a new location! If you need assistance during this time, please contact our Eugene office at 541-686-7696.
The office will reopen on Wednesday, April 1 at their new location: Douglas County Courthouse, 1036 SE Douglas Ave., Room 203, Roseburg. The new phone number is 541-957-2020.
Adobe Reader, or equivalent, is required to view PDF files. Click the "Get Adobe Reader" image to get a free download of the reader from Adobe.
Are you sure you would like to leave?
You are currently running an old version of IE, please upgrade for better performance.


text:
Hardening off
I have a number of things started from seed, grown in my basement under a combination of MH and HPS lights. They were all doing supurb, so last weekend I moved them to an unheated Greenhouse that is built with polycarbinate siding an roofing. It's double panel poly. The plants receive sunlight filtered through that poly. My plan was to let them acclimate in the GH for two weeks, then transplant them.
In the GH, thye are getting sunlight, but it's filtered by the polycarb. The location does not get full noonday sun, just moring and late afternoon.
Will these plants need to be hardened off to the "real" sun, or is the GH experience enough? Last year I skipped the GH step and moved directly to hardening off in the shade and for limited time. It worked pretty good, but I am now using the GH as a secondary step for plants before real outdoors.
This worked well for onions, shallots and leeks. They all were born under the lights, moved to the GH, then put out in the sun. They did not get any hardening time between GH and full sun, but it was late March, early April and the sun angle and intensity were less than now.
So, tomatoes, peppers, vines? GH or let em out to play?
I'd harden them off. As long as you gradually acclimate them to sunlight, the greenhouse is probably an unnecessary step. But don't move them straight from lights to full sun.
Another option is to plant them out but make a little open-sided "tent" over the seedlings using a bedsheet or row cover, to filter the sunlight for a week or two. That worked well for me and was less work than moving the plants around.
One vote for and one against. So, I guess it's ok that I'm questioning this myself.
My greenhouse is not used so much as a hardening off phase; it's more about space issues. My grow room, with 4 HID is about 75 sq ft of space and my greenhouse has about the same. So when it's warm enough for a particular crop to go out, I move it and start the next round of little ones.
I'm in Zone 5 (close to zone 6), so last weekend I moved Tomatoes and Artichokes out. Next weekend, it will be peppers.
I think I may try an experiment and harden off a few and plant a few and see what the difference is.
I would think the hardening off process could be sped up after the GH. Try one plant. Start with 4 hours sun the first day, 8 the second, morning and late afternoon. I think that's all they'll need. Personally I'd stick one out in full sun just to see. I don't have a GH but I harden my plants off in 4 days starting with 3 hours in the morning and move them further away from the house each day until they get sun from 9am to 4pm then they are ready. I start under overdriven fluorescents which aren't as good for preparing them for sun as your MH.
The one aspect the GH may not address is the wind. Plants not conditioned to it would have been snapped in two by the winds we had yesterday and it wasn't even storming! I have an oscillating fan in mine that runs 24/7 and none of mine suffered any damage.
I didn't harden off pepper seedlings that I started under an HPS fixture and they did fine being transplanted immediately. I also put brassica seedlings started under a fluorescent fixture under the HPS bulb for a couple days prior to transplanting and they did ok.
When I move my tomato and pepper plants from inside under fluorescent lights to my greenhouse, I cover them with a laundry basket for two days, unless very cloudy, then remove the basket.
When the greenhouse gets full, the oldest are moved to an outdoor shelter I built from old storm doors and a sliding patio door screen. This protects them from hail and strong wind. There, I partially shade them for two more days with a section of wooden slat fencing that reduces their sun exposure by about 50%.
Then, they go to the garden.


text:
What are you listening to right now? V3
WWE WrestleMania 31 in 720p HD
Ask a Verified Uploader Here
katpirat3 Uploads
Official Software Request Thread
Please request ebooks and audio books here. V9
Happy Torrents Day 2015!
Technical maintenance (March, 17)
Kickass.to domain back
Enjoy Coca Cola!
Do I got a question for you!
The Invisible Hand
I had a black dog, his name was depression
london lynn 1080p
tony 10 avi
the vow
wolverine anoxmous
the hometown of legends
tomb raider 2001
gravity 2013
the mentalist season 5 complete


text:
Why is it that whenever the Fighter is talked about, someone always brings up "oh they should be simple and easy to use so that new players can learn quickly".
I've been playing D&D since 2e and let me tell WotC something. I have never ran across a person whose first character is a fighter by choice (sometimes they were forced into it). I've seen time and again where the new person picks the more interesting option (cleric or wizard types) over that of the fighter.
And since the Fighter plays completely different than casters, why is it the goal to let people learn on the weaker and less versatile class. Won't they still be overwealmed when they level up to the big leagues? (Casters). Heck, doesn't it stand to reason that if you give the new person something bland they will get a negative impression of your product and stop using it? What if Pepsi, in order to get new customers, watered down their pop and gave it to people to taste test... I know I wouldn:'t come back to pepsi
How about we stop this nonsense and give interesting and useful things to the fighter? Expertise dice is a fantastic idea that has huge potential. I know this is a scary thought but pandering to the lowest common denominator never tends to work out.
I also would like to know why Fighters (and other mundanes) are stuck in low fantasy levels of play from level 1 to 20 when casters get to play in high fantasy. What is WotC afraid of? Why can't we allow the mundanes to walk with the big kids? D&D is a fantasy game and yet mundanes are, for the most part, limited to what people could possibly do in real life. I say bull crap, I don't want to play a fantasy game when to actually be a fantasy character I have to play a caster.
Give me extraordinary or give me nothing. Don't waste time on this same stuff that is already out there. I might as well just cut out all the mundanes because they give me nothing i cant do with a caster. Be bold, be daring, and be innovating. So far from what I've seen WotC is a quiet little 1/4 HD mouse that doesn't want to become the giant it once was or the giant that we want.
Rise up or forever be lost in the shadows of your competition.


text:
Obamas Embark on First Africa Tour
The first family will commence a lengthy trip to Senegal, South Africa and Tanzania on June 26. President Obama is slated to address key topics, like U.S. investment in and trade with Africa, while the first lady will continue her focus on youth, women and girls.
(Photo: AP Photo/Michael Probst)
World Lens: 500 Nigerian Civilians Allegedly Abducted03/25/2015
World Lens: Super Tropical Cyclone Ravages Pacific Nation03/16/2015
A Timeline of Recent Boko Haram Attacks03/11/2015
World Lens: Dozens Killed by Teen Suicide Bomber in Nigeria03/11/2015
World Lens: Netflix Pays Millions to Stream African War Film03/05/2015
Global Week in Review: Iran, US Talk Historic Nuclear Deal02/23/2015
World Lens: At Least 16 Dead in Haiti Carnival Accident02/17/2015
World Lens: Boko Haram Suspected of Explosion in Nigeria02/12/2015
World Lens: Boko Haram Kills at Least 90 in Cameroon02/05/2015
Google Doodles Celebrating People of Color02/02/2015
World Lens: Deadly Blast Destroys Mexico Maternity Hospital01/29/2015
Haiti Uncovered: 6 Photos of the Island's Cuisine01/27/2015
World Lens: Ebola Vaccine Study Set to Begin in Liberia01/23/2015
World Lens: Malian Hero of Paris Attack Awarded01/16/2015
12 Things You Must Know When Vacationing in Abu Dhabi01/13/2015
Global Week in Review: Paris Mass Shooting Suspects Killed01/09/2015
World Lens: Dozens Die at New Year's Eve Stampede01/02/2015
Global Week in Review: Africa's Growing Middle Class12/24/2014
Global Week in Review: Thousands Killed in South Sudan12/16/2014
Global Week in Review: Haiti Protests for Elections12/09/2014
Kenyan #MyDressMyChoice Campaign Spotlights Women's Rights12/04/2014
What We Know About the Nigerian Abductions11/21/2014
Haiti's Bull Fighting Culture Brings Fortune to Some11/19/2014
World Lens: Black Pete Protests Result in 90 Arrests11/18/2014
Global Week in Review: Plane Crash Kills Bahamas Pastor11/11/2014
Global Week in Review: WHO to Pick New Leader for Africa11/05/2014
Global Week in Review: Ebola Cases Surpass 10,00010/28/2014
Global Week in Review: Dozens More Nigerian Girls Kidnapped10/23/2014
Global Week in Review: Delayed Elections Amid Crisis in CAR10/07/2014
Global Week in Review: Boko Haram Leader Threatens Nigeria09/30/2014
Global Week in Review: Ebola Deaths Could Reach 1 Million09/25/2014
Swallowed by the Sea: A Look at Recent Migrant Ship Deaths09/17/2014
Global Week in Review: Human Zoo Exhibit Called Racist09/15/2014
A Look at What's Happening in Ukraine09/05/2014
What You Need to Know About the Israel-Hamas Crisis08/28/2014
Global Week in Review: Death Toll Rises in Ebola Epidemic08/26/2014
News Sasha and Malia Obama: Elevating... Clip (3/17/2015)
BET News Remembering Selma 50 Years Later Clip (3/7/2015)
BET News Obama: Because of Them, The... Clip (3/7/2015)
Celebrities Sigourney Weaver Responds to... Clip (3/5/2015)
BET Experience Michelle Obama Shouts Out BET! Clip (3/3/2015)
Wendy Williams Highlights: Kenya Moore Chats... Clip (2/3/2015)
B* Real Rita Ora Serves Vintage... Clip (1/30/2015)
News Obama's Swag On Display During... Clip (1/23/2015)
News What Should Obama Talk About in... Clip (1/16/2015)
BET News Free College Tuition Could Be... Clip (1/15/2015)
BET News North Korea's Racist Rant... Clip (12/30/2014)
BET News Top Ten Hashtags of 2014 Clip (12/23/2014)
BET News The Killings That Pricked Our... Clip (12/23/2014)
BET News Special Special: Justice for Us: A BET... Full Episode (12/10/2014)
BET News Special Special: Justice for Us: A BET... Full Episode (12/10/2014)
BET News Special BET News Presents: A... Full Episode (12/8/2014)
News President Obama Discusses Modern... Clip (12/7/2014)
News Stevie Wonder and Charlie... Clip (11/26/2014)
News No Indictment for Michael... Clip (11/24/2014)
Celebrities Kenya Moore Compares Atlanta... Clip (11/18/2014)
Will These Black Politicos Help Clinton Make History? 03/27/2015
Nigerian Military Claim to Destroy Boko Haram Headquarters 03/27/2015
Black Women Call on McConnell to Hold a Vote on Lynch 03/26/2015
World Lens: 500 Nigerian Civilians Allegedly Abducted 03/25/2015
Fired Univision Host Claims the First Lady Never Complained 03/25/2015
Raven-Symoné Says First Lady Looks Like a 'Little Cat' 03/24/2015
Michelle Obama Goes Global for Let Girls Learn Initiative 03/23/2015
Obama Hosts 2015 Student Film Festival 03/21/2015
Michelle Obama: Buzzing All Over the World 03/20/2015
Raven-Symoné Not Apologizing for Comparing People to Animals 03/20/2015
Poll: Public Unaware Obama's Health Law Is Again in Jeopardy 03/19/2015
Two More Aid Workers Flown Back to US For Ebola Monitoring 03/19/2015
Raven-Symoné Talks Univision Host's Michelle Obama Comment 03/17/2015
DuVernay Says Hollywood Not Lining Up for Black Protagonists 03/15/2015
Arrest Made in Shooting of Two Police Officers in Ferguson 03/15/2015
Shooting of Two Police Officers Sparks Anger in Obama 03/13/2015
Civil Rights Leader Rev. Willie T. Barrow Dies at Age 90 03/12/2015
When the Right Wing Attacks Music Stars 03/12/2015


text:
Horse Racing – The Musical [Video]
Listen as this announcer practically sings the call of this horse race.
Listen as this announcer practically sings the call of this horse race.
Here's a look at some notable birthdays being celebrated today:
David Lee Roth
Age: 57
Occupation: Singer and Songwriter
Known For: His party-boy persona, being the on-again, off-again lead singer of the band Van Halen, hits with them such as 'You Really Got Me,' 'Dance the Night Away,' and 'Panama,' solo hits such as 'Just a Gigolo,' and recently reuniting with Van Halen for a new album
On Sunday at Talladega, defending NASCAR Sprint Cup champ Tony Stewart set off a 25-car wreck, which somehow Matt Kenseth avoided, allowing him to come away with the win under caution. Fortunately this huge pile-up didn't lead to any infield confrontations, but quite often they do.
The NASCAR circuit has produced its fair share of high-profile feuds over the years (many involving Stewart). Most of the time, these disputes are settled with a few choice words and some mild jostling. On the other hand, it's never a huge surprise when two drivers come to blows after trading paint. Here's a look at a few of NASCAR's most memorable confrontations:
The pundits who follow NASCAR were calling last weeks race at the Monster Mile in Dover Delaware, "Separation Sunday". Those same pundits were calling the race at Talladega "Bring 'em Back Together Sunday". The
From 1959 to 1992, NASCAR legend Richard Petty won 200 races, setting a career record that is pretty much unmatchable for active drivers. Still, today's Sprint Cup racers compose arguably the strongest field of competition that the sport has ever known.
While stars such as Jeff Gordon and Jimmie Johnson may never reach Petty's incredible peak, they have managed to accomplish feats that eluded even The King. Let's take a look at the five winningest drivers on today's Sprint Cup circuit:
The best professional golfers from the US and Europe are in Chicago to duel for the Ryder Cup, while Major League Baseball winds down its regular season. Here's what to watch this weekend:
Many professional athletes have pre-competition rituals or quirks, but NASCAR driver Tony Stewart has one of the strangest ones we've heard of.
And week one in the Chase for the Sprint Cup goes to ... Brad Keselowski, kicking off the 10-week contest to determine NASCAR's 2012 champion.
Here's a look at some notable birthdays being celebrated today:
Constantine Maroulis
Age: 37
Occupation: Singer and Actor
Known For: Placing sixth in the fourth season of 'American Idol,' and his role in the musical 'Rock of Ages.'
The upcoming sports calendar features several key battles between ranked college football teams, the beginning of the NASCAR Chase for the Cup and Week 2 of the NFL. Here's what to watch this weekend:


text:
As the sun set over Sydney, about 100 people gathered to remember those who died because of asbestos-related illness.
Scooped by
Linda Reinstein
onto Asbestos and Mesothelioma World News
You are the content you publish.
Sign up with Facebook Sign up with Twitter
I don't have a Facebook or a Twitter account
Start a free trial of Scoop.it Business
Your new post is loading...
Your new post is loading...


text:
LYFE Kitchen in Culver City
Wednesday, April 17, 2013 | 2 years ago
Enter LYFE Kitchen, the first part of which stands for "love your food every day." LYFE opened in Culver City earlier this year with the noble aspiration of pioneering a new kind of franchise, one that serves healthy, affordable, fast-ish food. Culver City is the second location to open (Palo Alto was the first), with more California locations in the works and an ambitious plan to open 250 locations within five years.
See Also: LYFE Kitchen Review: This Would-Be Chain's Heart Is in the Right Place, But Its Food Isn't
View All
More Restaurants Slideshows
Le Comptoir Serves Luxury and Austerity7 days ago
Love & Salt's Mood-Lifting Cuisine14 days ago
L.A. Weekly's "The Essentials" Food & Wine Event 201521 days ago
Redbird Is a Welcome Addition to Downtown21 days ago
Beauty and Bites at Vegan Street Fair Los Angeles27 days ago
Eagle Rock Brewery Public House Goes Beyond Brewpub1 month ago


text:
EF 70-200mm f/4L USM
High-performance, L-series telephoto zoom lens combining light weight and compactness with an f/4 maximum aperture. Inner focusing and the ring USM enable quick and quiet autofocusing. Also, a circular polarizing filter can be attached and used without difficulty because the front lens element does not rotate during focusing. The tripod collar (sold separately) is the same one used with the EF 300mm f/4L USM.
Sample Images
Offer valid March 9, 2015 12:00 a.m. through March 31, 2015 11:59 p.m. ET. Offer valid in U.S.A. only, and void where prohibited, taxed or restricted. Availability of certain items may be limited. Limit of ten of any one item per order, subject to availability. Offer not valid on bulk orders. Order will be shipped to a street address in the 50 United States or the District of Columbia only. Dealers, distributors and other resellers are not eligible for this offer. Offer subject to Canon Online Store Terms of Sale.


text:
SNEAKER SEASON. These retro white-accented pairs are more on-trend today than they've ever been. The perfect finishing touch to almost any outfit, … [Read more...]
Essentials: Ooze Street-Savvy Attitude with White-Accented Sneakers
Exclusive Feature: Model Aurelien Muller Talks About Getting Fit and Taking Over David Gandy
FRENCH FLAIR. Classically handsome Aurelien Muller was scouted on his first day working in the fashion capital of Paris. Here's what happened … [Read more...]
Fashion Spread: The Most Comfortable Casual Wear in "Into the Woods"
INTO THE WOODS. As the year draws to a close, get off the grid and back to the nature, and live the adventure in the most comfortable casual … [Read more...]
Column: Milan Fashion Week Fall/Winter 2014/2015
SARTORIAL CHAMPIONS. Menswear luminaries in Milan stick to what they know best while gearing up for unpredictable future trends Etro's … [Read more...]
Feature: 5 Top Male Models to Know
DANDY DOMINATION. Male models are no longer mere props to female models and have carved a niche for themselves on and off the runways. DA MAN … [Read more...]
Exclusive Feature: Ryan Burns
BURN NOTICE. Ryan Burns has been in the modeling game some time yet remains at the very top Outfit by Calvin Klein Collection Outfit by … [Read more...]


text:
Sending messages to non followers
Hi Folks – is there a way to send our private messages to other members who are not following you?
On the same note, is the a pluging which can replace BP messaging system that you can think of?
- The topic 'Sending messages to non followers' is closed to new replies.


text:
content violation
You can write your comment.
We detected this website has 29 keywords. Keywords should not contain more than 260 characters so we recommend reducing keywords length for this website.
It is better to have a validated background for any website to be accesible from more browsers. This website has 0 error(s) and 0 warning(s). It is perfect that it doesn't have any xhtml errors.
Most people tend to type simple phrases on search engines instead of typing url on the address bar to access any website. Unfortunately we couldn't get search query data for this website yet and continuing our researches.
printecopy.es domain expires on 4/1/2015. If you are interested in this domain, you can contact the owner or wait for 0 days.
Title of this website is . And it has CISS,CISS con chip,EPSON,CANON,HP,BROTHER,toner,kits recarga,chip,chip reseteador,polvo,polvo toner,recargable,tinta,tinta canon,tinta hp,tinta epson,tinta brother,toner recargable,cartucho,cartucho i... keywords. A short summary for this page is
Ahorra hasta el 90% en impresiones con CISS (sistema continuo de tinta), no cambies m�s de cartuchos.Tambi�n CISS con chips resetadores,tinta para CISS,toners�. We found out encoding of this website is .
We located it in 126.96.36.199,
This domain has been created on 4/1/2015 and its latest update is in 4/1/2015. Domain will be expired on 4/1/2015. Nameservers are and . Found these information on whoIs server . Domain registrar is .
personalloanfinder.com.au is ranked #308011in the world. low doc personal loan is the most searched query for this domain.
personalloans.com.au is ranked #1013881in the world. loan application rejected mshwari is the most searched query for this domain.
petals-exchange.com is ranked #3569396in the world. petals exchange is the most searched query for this domain.
peoplehub.com is ranked #565191in the world. peoplehub.com is the most searched query for this domain.
peugeot.com.au is ranked #449562in the world. نوعية زيتون pdf is the most searched query for this domain.
perpetualdatasolutions.com.au is ranked #1269423in the world. perpetual data solutions is the most searched query for this domain.
petstock.com.au is ranked #516906in the world. petstock pty ltd is the most searched query for this domain.
performanceshop.com.au is ranked #845153in the world. inverter making noise is the most searched query for this domain.
perthweb.com.au is ranked #1405621in the world. ecommerce solutions perth is the most searched query for this domain.
peterstevens.com.au is ranked #836186in the world. peter stevens is the most searched query for this domain.
Country Idea is simply based on websites' distrubutions on countries. We help people to get an idea of which websites are popular in which countries, which nations look for what on internet, general information about the country of any website belongs and many little details. Users can also see which site is more successful in which country by comparing essential data such as Alexa Rank, Google Page Rank, etc.
We provide a variety of surveys for our users and save answers to our database. This gives us a unique new statistic of people preferences distributed on countries. We try to collect data in order to understand people better, such as life styles, eating and drinking habbits, thoughts about other countries and other websites belong to other countries.