text:
A rapid method for the quantitation of various live cell types is described. This new cell fluorescence method correlates with other methods of enumerating cells such as the standard plate count, the methylene blue method and the slide viability technique. The method is particularly useful in several applications such as: a) quantitating bacteria in milk, yogurt, cheese, meat and other foods, b) quantitating yeast cells in brewing, fermentation and bread making, c) quantitating mammalian cells in research, food and clinical settings. The method is especially useful when both total and viable cell counts are required such as in the brewing industry. The method can also be employed to determine the metabolic activity of cells in a sample. The apparatus, device, and/or system used for cell quantitation is also disclosed.
1. A method for detecting metabolic activity in viable yeast cells in industrial fermentation applications, comprising:
(a) providing a sample containing yeast cells,
(b) contacting said sample containing yeast cells with 1) a molecule or dye that is altered by metabolically active viable yeast cells into a detectably fluorescent form, wherein the molecule or dye comprises fluorescein diacetate or OREGON GREEN™ (2′,7′ difluorofluorescein), and 2) a compound that increases the rate of uptake of the molecule or dye into the yeast cells or speeds up the rate of conversion of the molecule or dye into a detectably fluorescent form inside said yeast cells;
(c) detecting fluorescence of the molecule or dye that is altered into a detectably fluorescent form by the metabolically active viable yeast cells in said sample, wherein the detection is performed by a fluorometer; and
(d) repeating step (c) over a time course, wherein a change in fluorescence over the time course indicates a change in metabolic activity of viable yeast cells.
2. The method of
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This application is a continuation application of U.S. patent application Ser. No. 12/340,044, filed Dec. 19, 2008, now U.S. Pat. No. 7,906,295; which is a divisional application of U.S. patent application Ser. No. 11/144,244, filed Jun. 2, 2005, now U.S. Pat. No. 7,527,924, issued May 5, 2009; which is a divisional application of U.S. patent application Ser. No. 09/912,266, filed Jul. 24, 2001, now abandoned; which claims priority to U.S. Provisional Application No. 60/220,298, filed Jul. 24, 2000.
1. Technical Field
The present invention relates to methods for the rapid quantitation of both viable and nonviable cells. More specifically, the invention involves incubating cells with a metabolically activated visible fluorescent dye and measuring the fluorescence generated by viable cells. Total cell populations (viable+nonviable) are separately determined by measuring the native UV fluorescence of the cells. The two fluorescence readings are directly related to the number of viable and nonviable cells. This permits the user to determine the percent viability of a mixed population of live and dead cells.
2. Description of the Related Art
The ability to quantify living cells is vitally important to the food, beverage, pharmaceutical, environmental, manufacturing and clinical industries. Several methods are currently employed by these industries to quantify prokaryotic and eukaryotic cells. These methods include, but are not limited to, the standard plate count, dye reduction and exclusion methods, electrometric techniques, microscopy, flow cytometry, bioluminescence and turbidity.
The standard plate count permits the quantitation of living cells (or clumps of cells) also known as colony forming units (cfu) when the cells are grown on the appropriate medium under optimal growth conditions (Microbial Ecology, Atlas, R. M. and Bartha, R., Addison Wesley, Longman, New York, 1998). Current standards of viable organism counts are often based on the standard plate count, particularly in the food industry. However, colony counts are difficult to interpret since bacteria often clump or form chains that can give rise to significantly inaccurate estimations of the total number of viable organisms in a sample. Also, bacteria, for example, can be in a "metabolically damaged" state and not form countable colonies on a given medium. This problem is more severe when selective media are used. Thus, the standard plate count does not provide a definitive count of viable cells in a sample, which may be very important for certain purposes. Given these factors, such testing also requires skilled technicians who can distinguish individual colony forming units and who can aid in selecting appropriate growth medium. Moreover, the technique is not useful when rapid determination of cell counts is required, since it often requires over 24 hours to obtain results.
Other tests, such as dye reduction tests, rely on the ability of cells to oxidize or reduce a particular dye (Harrington, 1998). Such methods are used to measure the activity of metabolically active organisms rather than provide a measure of the total number of viable cells in a sample. Dyes, such as methylene blue, coupled with microscopic counting, are routinely employed to determine the relative number of microorganisms. The technique is widely employed but nevertheless suffers from factors that must be held constant during the assay, e.g., medium used, chemical conditions, temperature and the types of cells being examined. Also, dye reduction tests that incorporate microscopic counting techniques require trained technical personnel and often depend on subjective interpretations.
Dye exclusion methods of cell quantitation depend on the living cells having the ability to pump the dye out of the cell and into the surrounding fluid medium. While the dye may enter the interior of both living and dead cells, dead cells are not capable of actively pumping the dye out under the conditions normally used. Dye exclusion is commonly employed to enumerate animal, fungal and yeast cells. It is a method requiring skill, correct timing and proper choice of dye. It is not applicable to certain microbes and it yields incorrect viable counts with stressed cells.
An accurate estimation of the number of viable yeast cells in a sample can be obtained by the slide viability technique (Gilliland, 1959). The yeast cells are suspended in a growth medium containing 6% gelatin and the suspension is placed in a hemacytometer slide. The cell suspension is incubated for approximately 20 hours and the numbers of micro colonies are counted. Cells that form micro colonies are viable and dead cells remain as single cells. This technique is considered by the brewing industry to be the most definitive test for counting the number of viable yeast cells. Unfortunately, the long incubation time makes it unacceptable as a routine method.
Microscopic techniques typically involve counting a dilution of cells on a calibrated microscopic grid, such as a hemacytometer. A recent improvement in this technique is the direct epifluorescent filter technique (DEFT) (Pettipher et al, 1989). In this technique, samples are filtered through a membrane filter that traps the cells to be counted. A fluorescent dye is attached to the cells, which are illuminated with ultraviolet light and counted. Unfortunately, the technique requires the use of an expensive microscope and a trained individual or an expensive automated system (Pettipher et al., 1989).
Yet other methods of quantitation use flow cytometry, which involves the differential fluorescent staining of cells suspended in a relatively clear fluid stream of low viscosity. The cell suspension is mixed with the fluorescent dye and illuminated in a flow cell by a laser or other light source. The labeled cells are automatically detected with the use of a fluorescence detector focused on the cells (Brailsford and Gatley, 1993 and Pinder et al., 1993). The technique requires, and is limited by, expensive equipment. Some flow cytometric devices have been used by the food and dairy industry, but their application has been limited by the high cost of instrumentation.
Bioluminescence has been routinely employed in the food sanitation industry to detect and quantify viable organisms and cells. The method involves the use of luciferin-luciferase to detect the presence of ATP (Harrington, 1998 and Griffith et al., 1994). When used to quantify cells, the technique depends on the assumption that there is a constant amount of ATP in a living cell. ATP levels vary in a single cell over more than two orders of magnitude, making this method a relatively inaccurate technique for the enumeration of viable organisms in a sample.
Turbidity of a liquid sample can also be measured as an indication of the concentration of cells due to the light scattering and absorbing qualities of suspended cells (Harrington, 1998). The method is old but it is still employed to estimate the bacterial concentration in a sample. The method is rapid and simple but is highly inaccurate since all cells, particles and substances, including non-living particulate matter, interfere with the interpretation of the results.
The present invention for the quantitation of both viable and nonviable cells is designed to overcome at least five problems that have been identified within the field. First, the new technology circumvents the need for training personnel in how to plate, grow and count viable cells from colonies on agar plates. It also eliminates nearly all training and maintenance costs associated with most of the other methods. Second, the invention substantially decreases the time needed to determine concentrations of cells such as yeast and bacteria. Under current methodologies, quantification requires from 24-72 hours (plate count and enrichment cultures), while the present invention permits accurate quantitation in less than 15 minutes. The methylene blue test is rapid; however, the accuracy is unacceptable for cultures that are less than 90% viable. The slide viability test is accurate for large viability ranges but the time required for results is not suitable for routine use. Third, the new test is accurate over wide ranges of viability and has precision similar to the slide viability test. Fourth, the instant invention offers substantial cost savings over existing methods of cell quantitation. Fifth, the invention permits the simultaneous determination of both viable and total cells in a sample. This allows the user to accurately establish the percent viability of a cell sample (the number of viable cells to total cells). Percent viability is a crucial measurement in many industries such as the dairy and beer brewing industries and is currently carried out by the methylene blue test.
The present invention generally provides methods, kits, and devices for detecting and quantitating the number and/or percentage of viable cells in a sample. In one aspect the invention provides a method for determining the percent viability of cells in a sample, comprising providing a sample containing said cells, detecting the total cell count, contacting said cells with molecule or dye that is detectably altered by enzymatic activity of a viable cell, detecting enzymatically altered dye or molecule, thereby detecting the number of viable cells and comparing the number of total cells with the number of viable cells thereby determining the percent viability.
In another aspect, a method for detecting viable cells is provided that comprises providing a sample containing cells, contacting said sample with a dye that diffuses or is transported into said cells and wherein said dye is detectably altered by enzymatic activity of a viable cell, thereby detecting viable cells in a sample.
Yet additional aspects of the present invention include methods for quantitating viable cells in a sample, comprising providing a sample containing said cells, contacting said cells with molecule or dye that is detectably altered by enzymatic activity of a viable cell, detecting enzymatically altered dye or molecule, thereby detecting the number of viable cells in said sample and obtaining a value therefrom and correlating the detected viable cell value with a standard value, thereby quantitating the viable cells in said sample.
Further aspects include methods for quantitating total and live cells in a sample, comprising measuring total fluorescence of cells in a sample and comparing to a standard value, thereby quantitating total cells in said sample; contacting a sample with a fluorescent dye that is metabolically altered by live cells; said dye having fluorescence properties that are measurably altered when modified by live cells, detecting the metabolic alteration of the dye thereby obtaining a measurement value and comparing said value to a standard value, thereby quantitating live cells in said sample.
Still other aspects of the present invention include methods for measuring the number of total and live yeast, bacteria or other cells in a sample, comprising measuring the native fluorescence of cells in suspension, contacting said cells with a dye that penetrates into the interior of yeast or bacteria and is metabolically modified to a measurable parameter by live cells, measuring the total fluorescence and fluorescence properties provided by the metabolic alteration of said sample and correlating said fluorescence to the number of total and live cells in said sample or a fraction of the sample and determining the percent viability of said sample.
In certain embodiments the cells may be of any origin such as bacteria, yeast, or mammalian. In related embodiments the total cell count is determined by a method selected from the group consisting of native UV absorption, turbidity testing, hemacytometer measurements, fluorescence, and dye exclusion.
In yet other embodiments, the enzymatic activity that alters the dye or molecule is esterase activity. In further embodiments the enzymatically altered dye or molecule is fluorescein diacetate or OREGON GREEN™.
Other embodiments include measurement by a device, such as by a fluorometer.
The invention also provides kits for quantifying yeast or bacteria, comprising a cell suspension solution, a cell penetrating dye, and instructions for detecting dye that correlates to hemacytometer counts, plate counts or other methods of counting viable cells.
In certain embodiments the kit includes a dye that is enzymatically and detectably altered following penetration of viable cells.
In certain aspects a kit for quantifying yeast or bacteria or mammalian cells is provided, comprising: a first container containing a first solution, a second solution containing a compound that penetrates cell membranes and is metabolized to a fluorescent dye or other detectable dye that is measurable, and instructions for using the same.
In other embodiments kits of the invention further comprise a means for mixing said first solution with a sample containing an unknown number of living cells and nonliving cells, means for concentrating the cells from the mixture of said first solution with said sample and removing solids from the remainder of said mixture, and measuring native fluorescence of cells in said solution.
In still yet other embodiments the kits may further comprise a means for mixing said second solution with said cells to form a second mixture, and means for illuminating the mixture of said second solution with said cells with excitation light and measuring fluorescence emitted by said mixture, and thereby determining the amount of metabolically modified dye present in the cells that is proportional to the number of viable cells in said second solution.
In other embodiments the kits may further comprise a third solution containing a compound or compounds that increase the rate of uptake of dye into cells or speed up the rate of conversion of the detectable fluorescent form of the dye inside said cells in second solution.
In another embodiment, a device comprising solid fluorescent material consisting of an adaptor and a compound that can be used to calibrate the instrumentation used for detecting fluorescence in the cells is disclosed.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings.
Briefly, the current invention describes novel methods that can be used to quantify live cells and total cells (total includes all cells in the sample, both viable and nonviable) such as yeast and bacteria. This allows the user to determine percent viability of the sample of cells. In one aspect, the instant invention comprises three steps: 1) determination of total cells, 2) determination of viable cells and 3) calculation of percent viability. In certain embodiments the total cells are determined by washing and incubating the cells in a solution and then measuring the native UV fluorescence of the cells in a fluorometer, thereby permitting the determination of total cell populations. Subsequently, the cells are incubated with a compound that can be metabolically converted to a visible fluorescent dye such as fluorescein diacetate, coupled with an inducer of esterase activity such as dequalinium acetate, and then the fluorescence is measured, thus permitting enumeration of viable cell populations. The fluorescent readings are correlated to standard counts such as hemacytometer counts or to the slide viability counts. The two fluorescence readings are directly related to the number of total and viable cells respectively. This permits the user to calculate the percent viability of a mixed population of live and dead cells.
As those of ordinary skill in the art can readily appreciate the present invention may be modified in certain ways to achieve the same result. In brief, the present invention utilizes one or more dyes or molecules that allow for the detection of all cells or total cells (e.g., yeast, bacteria, mammalian, etc.) in a sample and the same or different one or more dyes that are metabolized/derivatized by the viable cells in the sample to allow detection of the viable cells. Accordingly, the percent viability can then be readily determined. As can be appreciated, substances such as detergent-like compounds, surfactants, solvents, or other compounds that affect membrane polarity, membrane fluidity, permeability, potential gradient, etc., may be added to the sample to increase the rate at which the molecule or dye enters the cells in order to speed the process.
Other variations that are within the scope of the present invention include adding compounds that affect membrane polarity to decrease the rate of "leakage" of the converted dye from the cells. Further, esterase enzyme inducing chemicals such as naphthalene or dequalinium acetate may be added to increase esterase activity in living cells. In addition, esterase activity may be increased by environmental factors such as heat. Furthermore, compounds other than fluorescein diacetate, such as Calcein AM, may also be used to detect metabolically active live cells.
The stability and shelf life of the fluorescein diacetate and other chemicals may be increased by the addition of antioxidants or similar preservatives or by dissolving the FDA into other solvents besides acetone or by other stabilizing methods such as lyophilization. The fluorescence detection apparatus used may be designed for microscopic, surface, internal, solution and non-suspension sample formats.
Also included within the context of the present invention is software that permits the user to interface the fluorescence instrumentation to a computer for direct calculation of percent viability and cell concentration or other data processing or recording formats.
Compounds such as hemoglobin may be utilized to reduce background in the sample. Rinsing the sample in a buffer solution and centrifuging or filtering or otherwise retaining the cells as they are washed can be used to remove any exogenous background fluorescence.
The differences between prokaryotes and eukaryotes may also utilized to assist detection. For example, such easily detectable differences include cell membrane receptors, lack of organelles in prokaryotes, or metabolic differences. These differences can be utilized to distinguish between prokaryotes and eukaryotes by using dyes that penetrate only mitochondria or nuclei for example, or to take advantage of membrane and metabolic differences in these two cell types. This will allow the user to count a specific prokaryote or a eukaryote in a mixture of cells that contains both types of cells. For example, one may determine if bacteria contaminate a yeast cell or blood cell population.
Other variations of the present invention include altering the pH of the reaction solutions to increase the sensitivity of the reaction. Further variations include changing concentrations of the solutes to increase or decrease the sensitivity of the reaction. Alterations to the solid standards may be utilized to increase the sensitivity and dynamic range of the assay. The viability assay may be used to measure overall health or metabolic or growth status of the cells, including the ability to withstand stress. The various steps of the tests may be used independently, e.g., to measure only total cells or only live cells.
Other methods of total cell determination may include using DNA binding dyes, protein stains, cell membrane stains, antibody coupled stains, lipid dyes or other methods of detecting total cells in a sample. Viable cells may also be quantified using other methods that distinguish between live and dead cells such as surface markers, DNA stains, protein stains, antibody coupled stains, lipid dyes or other methods of detecting viable cells in a sample.
The solid standard can be made out of other materials such as, but not limited to, plastic, or by embedding chemicals such as fluorescein in a solid matrix of epoxy, acrylic, polyacrylamide or agarose or by coating a material like plastic with said chemical. Other chemicals, which have excitation and emission wavelengths in the range of the dyes or cells used to carry out the invention, could be used.
The instrument can be calibrated with solutions containing fluorescent chemicals such as fluorescein or OREGON GREEN™. Other configurations of the solid standard can be employed. The adaptor can be constructed to fit the type and make of instrument used to carry out the method. Different concentrations of the reagents may be used to carry out the method. Other methods of mixing and or concentrating samples may be used. The wash steps may also be eliminated, thus simplifying the procedure.
Yet further variations of the present invention include, but are not limited to, the use of an incubator to control the temperature of the dye conversion in the cells. Such incubation can take place in a plate counter or vial heater of some kind Further, the samples may be arranged in an array format to allow high throughput detection.
The methods and kits of the present invention allow for the determination of the number of active cells by measuring the rate of conversion of dye by the cells. The number and activity of said cells may be determined without reaching the reaction endpoint.
The methodology has obvious application in determining the activity of yeast or bacteria in industrial fermentation applications. Thus, the methods and kits can be used to predict the number of cells required to carry out fermentation based on viable cells rather than on total cells.
As those of ordinary skill in the art can readily appreciate, the instant invention can be carried out in a single vessel and solution.
The instant invention also has applicability in assessing the activity, vitality, or number of cells under various storage conditions, comparing the metabolic activity of different cells, developing pitching rate charts for fermentation applications, and use of the method as a self-contained laboratory.
The present invention provides methods, kits and apparatuses for simple dye associated quantitation that allows one to inexpensively determine total cell counts and viable cell counts in a particular sample. An individual of ordinary skill in the art will readily appreciate that alternatives to the steps herein described for quantitating cells may be used and are encompassed herein. Accordingly, all alternatives will use a kit or method wherein a dye is utilized to stain cells and a method is used to detect or quantify the dye. One key aspect of this invention is its ability to simultaneously determine total cells and live cells in a sample in short times compared to standard methods. In preferred embodiments, detection is completed in less than 4 hours, in others in less then about 3 hours, and yet further, in less than about 2 hours, while in specific embodiments, detection is completed in less than about 1 hour, less than about 45 minutes, less than about 30 minutes, less than about 15 minutes, and less than about 10 minutes at low cost.
All patents, patent applications and references cited herein are incorporated in their entirety. Accordingly, incorporated herein by reference are U.S. Pat. Nos. 5,437,980; 5,563,070; 5,582,984; 5,658,751; 5,436,134; 5,939,282; 4,783,401; 3,586,859.
The following shows examples of solutions, volumes and concentrations that can be used to carry out the invention. Other concentrations and volumes and different buffers with different pH values may be used. The samples and reaction solutions may be provided in any volume necessary for detection, however, in the present embodiment, small volumes (less than 1 ml) are utilized such that reagents and sample amounts are kept to a minimum. The volumes and concentrations to be utilized are any that are convenient to the practitioner of the methodology. In certain embodiments, the sample and reagent amounts range from 1 to 10,000 micro liters.
The reader used in the present embodiment is a "picofluor" hand-held fluorometer from Turner Designs, Sunnyvale, Calif. 94086. Any fluorometer that can measure fluorescence at specific wavelengths for detection of the dyes or cells can be employed in the invention. Ideally, the fluorometer can switch back and forth from visible (486 nm excitation with a 10 nm bandwidth and 550 emission with a 10 nm bandwidth) and UV (300-400 nm excitation and 410-700 nm emission) modes without changing filters or making other adjustments; however, this is not necessary to carry out the methodology. Other wavelengths may be used in combination with different dye types or cell types. The wavelength chosen will be dependent on the dye or cell type chosen for staining.
The instrument needs to be calibrated in a range that permits quantitation of cells. Calibration can be carried out with solutions such as fluorescein or OREGON GREEN™ or with the use of solid standards as described below.
Solid Calibration Standards:
I. Calibration Using Solid Standards
To calibrate the instrument in "A" mode (UV): Standard Value=500
To calibrate the instrument in "B" mode (Visible): Standard Value=500
Total cell counts can be determined by a variety of methodologies, including using the fluorometer noted above. UV operation mode is selected. A fixed volume (200 microliters) of Cell preparation solution is added to the glass sample vial. 5 microliters of sample (yeast cells) is then added to the Cell preparation solution in the vial and centrifuged for about 30 seconds to sediment the cell pellet. The cell pellet is resuspended in about 100 microliters of Cell preparation solution to the sample vial. The sample vial is then read in fluorometer. See
Live cells are quantitated utilizing a dye that is detectably altered by an intracellular enzyme. For example, the fluorometer noted above is set in visible mode and a fixed volume (200 microliters) of Cell preparation solution is added to the sample vial. 5 microliters of sample is added to the solution A in the sample vial and centrifuged for 30 seconds. 100 microliters of Suspension solution is added to the sample vial and 5 microliters of Reaction solution is added to the sample vial. The sample is mixed and placed in the reader and fluorescence determined at time zero and again at 15 minutes. This is the value that will be compared to the Easy Count correlation chart for conversion to cells/ml. See
Yeast performance is critical to the development of quality beer. For this reason, methods of yeast analysis are an important element of the brewing process. Traditional methods including hemacytometer counting and methylene blue staining are rapid, but inaccurate and unreliable. Slide culture is an accurate measure of yeast viability, but requires a lengthy incubation period of 18 to 24 hours. As an alternative, the fluorometric assay described above is based on the metabolic activity of the yeast culture to provide brewers with a rapid and accurate estimation of active cell number. This method was compared to the hemacytometer counting technique as an estimation of cell number, and to both methylene blue staining and slide culture as measures of vitality and prediction of fermentation performance. The inventive method correlated to the hemacytometer, methylene blue, and slide culture with R2 values of 0.985, 0.987, and 0.962 respectively, P<0.0001. An error analysis was carried out by the inventive methods, hemacytometer and methylene blue staining techniques for multiple operators performing the tests. Thus, the present invention could be used to determine correct pitching rates, monitor fermentation and propagation, and for other applications involving cell quantitation.
All yeast cultures were obtained from Wyeast Laboratories, Mt. Hood, Oreg. Yeast samples for the experiments comparing the hemacytometer, methylene blue staining, and slide culture to the inventive methods were a 1084 strain of Saccharomyces cerevisiae. Yeast cultures tested during laboratory scale fermentations were strain 1968. Brewery scale fermentations were performed using yeast strain 1056.
Hemacytometer Counts
Hemacytometer counts were performed according to the ASBC method (6,8). Samples were removed from a slurry with an initial concentration of 198 million cells per ml, as determined by hemacytometer count, and diluted in spent wort to maintain cell integrity. Each dilution was counted in the hemacytometer as well as measured using the present method. Experiments were carried out in triplicate.
Methylene Blue Staining
Methylene blue staining was performed according to the ASBC method (6,8). Samples were removed from a slurry with an initial concentration of 198 million cells per ml, as determined by hemacytometer count, and diluted in spent wort to maintain cell integrity. Each dilution was stained and counted in a hemacytometer, as well as measured using the present method. Hemacytometer counts were corrected for viability according to the staining results. Experiments were carried out in triplicate.
Slide Culture
Slide culture was performed according to a modified version of the protocol for preparation of slide cultures for the examination of yeast and mold (5). Ten ml of yeast strain 1028 at a concentration of 433 million cells per milliliter, as determined by a hemacytometer count, were placed into a 43° C. water bath. Aliquots were removed at time intervals 0, 2, 4, 6, 8, 10, 15, 20, 30, and 40 minutes. Each was tested using the inventive method. Measurements were taken in triplicate and averaged. Slide culture samples were diluted 1:100 into wort containing 6% gelatin. 10 μl of the sample were then placed on a micro slide, covered and sealed with petroleum jelly. Each slide was incubated for 20 hours at 18° C. before microscopic examination (Microscope model, Leica DMLB). Viability was determined with the assumption that living cells had formed micro colonies, while nonviable cells remained single.
Determination of Active Cell Number Using the Instant Invention
The inventive method for determining total active cell number is based on the metabolic activity of the yeast culture. The technology involves exposing cells to proprietary chemicals that enter cells through diffusion. These molecules are converted to a fluorescent form by metabolically active cells. This fluorescent signal is quantified in a handheld battery operated fluorometer model GP320 GenPrime Inc, Spokane, Wash. The protocol is as follows: 50 μl of yeast sample was added to 500 μl of cell prep solution in a 1 ml glass test cuvette. 50 μl of dye solution was added; the cuvette was capped, and incubated for 5 minutes. After incubation, the cuvette was shaken, and the fluorescent signal quantitated in the GP320. These values were compared to the hemacytometer and methylene blue staining methods by performing tests with the inventive method on the diluted samples from these experiments. Readings were taken in triplicate and averaged. These relationships were analyzed by linear regression using Statview, SAS institute, Cary N.C.
Fermentation Tracking
Laboratory Scale: Laboratory scale fermentation tracking was carried out in a 300 ml flask by inoculating 150 ml wort with 5 ml yeast strain 1968, with an initial concentration of 420 million cells per ml, and monitoring growth using a hemacytometer and the inventive methods. Cells were grown at room temperature (21° C.). Samples were taken every 45 minutes for 5.25 hours and then periodically over the next 48 hours.
Brewery Scale: Brewery Scale fermentation tracking was carried out during a typical fermentation cycle, at the Steam Plant Grill, Spokane, Wash. 99201. Hemacytometer counts and corresponding readings using the instant invention were made daily for 13 days beginning immediately following pitching.
Percent error between operators was determined for the inventive method "Easy Count" method, hemacytometer counts, and the methylene blue staining method. Error analysis was performed using Microsoft Excel.
Hemacytometer: Three operators performed hemacytometer analysis of a yeast strain 1028 slurry according to the ASBC method. Each operator prepared and measured 15 samples. Results were averaged for each operator, and error between operators was calculated.
Methylene Blue: The 15 hemacytometer samples from above were stained with methylene blue according to the ASBC method. Each of the three operators counted stained cells for each sample. Results were averaged for each operator, and error between operators was calculated.
Easy Count: Easy Count tests were performed on 15 replicate samples by each of the three operators. Results were averaged for each operator, and error between operators was calculated.
Hemacytometer Counts
Methylene Blue Staining
These results suggest that the Easy Count can be used to accurately predict active cell number. Using the results of the correlation, it is possible for the brewer to accurately determine the correct pitching rate using the Easy Count method based on 1 million active cells per ml per degree plato of wort. Additionally, the method can be used to monitor fermentation, propagation, and for other applications involving the quantitation of cells.
Slide Culture
A linear relationship was found between the Easy Count and slide culture for yeast viability, as shown in
The correlation to slide culture confirms that the Easy Count only measures active cells, since the total number of cells in this experiment remains constant.
Fermentation Tracking
Cell growth was measured during laboratory and brewery scale fermentations using both the ASBC method for hemacytometer counts, and the Easy Count method.
Results from the experiments were averaged for each operator as shown in Table 1. Percent error between operators was calculated by dividing the standard deviation of the mean by the mean, and multiplying the result by 100. Easy Count reported significantly lower error between operators than the other methods. These results are graphed in
Results of the error experiments confirm previous research reporting the inaccuracies of hemacytometer counts and methylene blue staining (1, 2, 3, 4, 7). The low error associated with the Easy Count method is an improvement on these traditional techniques.
Percent error is of particular importance to the brewer due to the exacerbation of inaccuracies in the calculation of cells/ml. For example, when calculating cells/ml from a hemacytometer count of 180 live cells and 15 dead cells (counting all 25 fields and using a 1:100 dilution), the result would be 180 million live cells/ml (180*100*10000) and 15 million dead cells/ml (15*100*10000). If the error between operators when performing the live cell test is 21%, then the live cell result could be between 142-218 million cells/ml, a difference of 76 million cells/ml. With a percent error of 28% between operators, the dead cell result could be between 11-19 million cells/ml. This could result in reported viabilities between 87% and 96% for the same sample. The Easy Count has much less error associated with its performance. A reading of 6000 in the Easy Count would be 197 million active cells/ml (see equation generated in
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From sexy tropical drinks to sophisticated and creative cocktails, this handy guide shares the secret cocktail recipes of top bartenders and mixologists at bars, restaurants and hotels around Mexico. You just might feel like you're on a Mexico beach vacation when you sip these divine creations! From the editor of LatinFlyer.com, the Latin America travel site.
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Newbe with no experience buys big boat
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Re: Newbe with no experience buys big boat
Just finished the book. I enjoyed it quite a bit.
Something about the way you wrote regarding the character of Joyce made her very real and almost familiar to me. Your interactions with her seem to have been very intense and I have the impression that she definitely left her mark on you. It would be very interesting to hear how she would tell the story of your trip. Have you ever heard her tell her version of the story? Not saying that as if there would be controversy, rather it would be fun to hear how two people perceive the same event in different ways.
I hope you continue to sail as it sounds like there is so much that you enjoy about sailing. There are plenty of intermediate places between "leaving it all behind and circumnavigating" and "not sailing at all."
Coastal cruising for weeks or months at a time is very rewarding with enough challenges to satisfy many sailors and with the benefit of never being far from internet, news papers, and all the convenience of shore life. Well, at least here on the east coast I find coastal cruising to be that way. My own long term cruising goals do not extend beyond the Virgin islands, at least not yet.
Hopefully you'll stick around sailnet, there are some good folks here, some world cruisers, many day and weekenders, and plenty of us coastal folks.
It's my happy pill
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Advantages of Piezoelectric Actuators in Haptic Feedback
Midé Technology has released a haptic evaluation kit to demonstrate how piezoelectric haptic actuators are able to provide a higher performance and richer tactile haptic feedback experience.
Medford, MA (PRWEB) July 31, 2013
Midé Technology has unveiled SHIVR™, its line of high performance, low-profile piezoelectric haptic actuators – ideal for phones, tablets and other handheld or body worn devices that require communication through the sense of touch.
Currently, magnetic actuators dominate the haptic feedback market; however, piezoelectric haptic actuators are an emerging technology that has many advantages over conventional magnetic actuators.
Piezoelectric haptic actuators are able to provide more than a simple buzz. They can create a richer haptic experience, as they allow single cycles, fast start-up, static deflection, and independent frequency, amplitude, and phase control capabilities – allowing the user to control the feel and intensity of the sensation.
The high performance and broad array of haptic feedback they provide are ideal for any application that requires a low profile feedback component. Additionally, piezos are low power, thin, lightweight, and non-magnetic, making them ideal for specialized bio-medical and industrial communication applications.
Midé's SHIVR piezoelectric haptic actuators share all of these advantages, plus the benefits of Midé's Piezo Protection Advantage.
Using flex-rigid-flex manufacturing techniques, high performance piezos are packed into a protective skin with pre-attached leads, making them hermetically sealed, electrically insulated, and robust.
They also have built-in electrical connections, providing the capability of creating multi-touch point arrays and a wide range of termination options – making them easy to integrate into any application.
Midé first started developing its haptic feedback actuators when it won a DARPA contract to develop a tactile actuator (tactor) for the purpose of supporting communication between land based soldiers in degraded audio and visual sensory environments. The solution was a stand-alone unit that can communicate independently, or be part of a body worn array of tactors.
Following the success of the tactor development, Midé is now launching its piezoelectric haptic actuators into the broader, but specialized, bio-medical, electronic gaming, and industrial markets.
Midé also offers the SHIVR HEK-200 Multi-Actuation Evaluation Kit; a complete kit which drives its piezoelectric haptic actuator in three different scenarios – direct actuation, bonded actuation and inertial actuation. The kit generates four pre-written haptic patterns and provides an optional user input.
Jared Keegan, a Senior Engineer at Midé and the SHIVR product manager stated, "We are very excited about the response we've already received regarding our haptic feedback evaluation kit. It's an educational tool that allows users to really get a feel for the different types of haptic feedback scenarios piezos are able to offer."
If you'd like more information about this topic, please visit our website (http://www.mide.com), or to schedule an interview with Jared Keegan, please call Jared at the contact information provided.
For the original version on PRWeb visit: http://www.prweb.com/releases/2013/7/prweb10986414.htm


text: Instagram / LeBron James LeBron James Will Return to Play for Cleveland Cavaliers By Chris Chaberski July 11, 2014 In a stunning move, the best basketball player on Earth, LeBron James, has decided to return to his hometown team, the Cleveland Cavaliers. Read More|Comment Category: News, Sports Tags: Cleveland Cavaliers, LeBron James Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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Los Angeles, CA
March 31, 2009 to April 2, 2009
pp: 557-558
DOI Bookmark: http://doi.ieeecomputersociety.org/10.1109/CSIE.2009.941
Formal grammars have been used extensively in the analysis and design of programming languages and compilers. This paper gives a brief introduction to the application of stochastic context free grammars (SCFGs) to model RNA. Then it briefly presents the parsing algorithm for RNA. Next it introduces the bottom-up LR parsing algorithm. Finally it proposes a table-based parsing algorithm for RNA. The proposed algorithm is an extension of the LR parsing algorithm.
Muhammed S. Al-Mulhem, "Table Based Parsing for RNA", CSIE, 2009, 2009 WRI World Congress on Computer Science and Information Engineering, CSIE, 2009 WRI World Congress on Computer Science and Information Engineering, CSIE 2009, pp. 557-558, doi:10.1109/CSIE.2009.941


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Dealing with hank-on-headsails
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My previous boat had hanked-on headsails, and I would drop it along the gunwale, inside the lifelines, and then pull it aft, so that it was flaked down in neat folds. Then I folded the aft 1/3 of the sail forward, and folded that over the forward 1/3 of the sail. The result was a fairly neat bundle, that was approximately 1/3 the length of the foot of the sail. Every time I folded it, it flaked a little differently, so I didn't get significant creases. The size of the bundle was too large to fit into the factory-supplied sailbag, so I made a bigger bag out of Sunbrella. Overnight, I would leave the bagged sail on deck. For longer periods of time, I stored the sail in a locker.
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Tell us your vacation horror stories
Published: Friday, June 4, 2010 at 12:18 p.m.
Last Modified: Thursday, June 10, 2010 at 3:33 p.m.
We take vacations to rest and get away from it all, but sometimes our vacations can turn into such horror stories that we find ourselves eager to return to work. As the summer season is upon us, we're asking readers to share their vacation horror stories for an upcoming feature. Send your stories to features editor David Moore at firstname.lastname@example.com, and include your name and contact information.
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.


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New York, February 14- Fres Oquendo, KO artist Curtis 'Showtime' Stevens to be there -- Broadcast Brothers John and Derek Tabacco started covering local sports on Staten Island over a decade ago with one goal: develop a sports show for die hard sport aficionados that want to know everything there is to know about sports.. The two Staten Island "paisans" not only accomplished their goal but went one step further creating sports programming and entertainment packed with beautiful women, celebrities and total Tabacco Mayhem!!! You can catch their next Valentine's Day broadcast live in the VIP section of the Grand Ballroom at the Manhattan Center on February 16, 2006 at 7PM. If you would like to meet the Locatestock girls and join in the Tabacco Mayhem contact DiBella Entertainment for tickets at 212-947-2577.
This year's first installment of Broadway Boxing will feature the return of heavyweight contender Fres Oquendo (24-3, 15KO's) as he challenges heavyweight veteran Daniel Bispo (16-2, 10 KO's) of Sao Paolo, Brazil in a 10 round contest. In his first co feature Curtis Stevens (10-0, 9KO's) will be showcased against Jose Spearman (29-9-4, 10 KO's) in an 8 round super middleweight bout that is sure to be Stevens toughest opponent to date.
This is only one of many entrepreneurial ventures that the Tabacco brothers are involved in. In 1998 the two founded Tori enterprises which serves as the parent company for all of their business enterprises. After operating successfully for five years the company branched out and locatestock.com was formed. Currently, the two brothers are in the developmental stages of production to launch their latest venture a new company called Tabacco Media and Entertainment. Tabacco Media and Entertainment will create 3 Tabacco TV channels that will stream over the internet providing viewers with continuous 24 hours of original programming. Tabacco TV and its subsidiaries are looking to become the industry leader in entertainment for delivering original television programming to homes across the world via the internet.
John Tabacco, the older of the two, graduated from St. John's University with a bachelor of science in finance. John is a thirty something, soft around the middle family man with three children who is determined to bring home the bacon while satiating his endless need to consume sporting events and sports programming while keeping his marriage out of divorce court. Derek Tabacco, the baby brother, is also a thirty something, single, athletic bachelor and womanizing legend- in his own mind- (only in his own mind).
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The storm that tore through parts of Okalahoma City and neighboring areas Monday is being called one of the worst tornadoes in history. David Greene talks to storm chaser Josh Wurman, who is the director of the Center for Severe Weather Research, and he studies tornadoes.
Source: NPR
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King Chair with Seat and Back Cushions
by Nova Solo
At A Glance:
Wrapped in striking woven rattan, the King chair features plush seat and back cushions that ensure a relaxed and comfortable seat. A casual, beach-house inspired style, the King chair features a rounded wingback design with bold, curved framing. Finished in a natural gray, this chair looks great overlooking the mountains or the ocean. Plush seat and back cushions create a comfortable and supportive place to kick back and enjoy friends, family, and nature.
- Overall Size: 49.25" h x 31.5" w x 36.25" d
- Natural grey color
- Kubu rattan material
- With back
- With arms
Materials & Measurements
Materials: Kubu Rattan
Measurements: 49.25" h x 31.5" w x 36.25" d


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We understand our selection of OccuNomix Tools can seem somewhat intimidating at first glance. For this reason we made sure to create this easy to use page! Our store brings together a huge amount of different product types from many manufacturers, and the various categories we offer help very different, but still demanding enthusiasts. Law enforcement and military professionals can browse through an abundance of options of riflescopes, red dot sights, reliable holsters and more, while laboratory technicians and researchers restock their lab consumables, upgrade their stereo microscopes, and outfit their astronomical observatories with the best new laboratory equipment. And our commitment to your full satisfaction does not stop at phenomenally low prices and an insane selection. We have a complete in-house customer service team in Northern Illinois and teams of highly-trained specialists who are happy to help as best they can. Our How-To Article Section also serves as a simple way to be certain you find just what you need, as more understanding of just how the gear you need works and how to use it will help you make a better informed buying decision. OpticsPlanet is your first retailer when looking for the best OccuNomix Tools in the world, so make this page a favorite and return frequently because we work day in, day out to expand our selection! Rest assured, when you shop at OpticsPlanet.com, your experience will be nothing short of excellent
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Coming soon: tune into The Outdoor Channel for the first-ever episode of the X Ring. Stay tuned for exact day and time.
The X-Ring is the center of the shooting world. It's where hunters aim and shooters are made. And at the core of this X-Ring is a dynamic duo adding a whole new dimension the Outdoor Channel lineup.
Former MLB baseball player and current television personality of the wildly-popular Buck Commander series, Matt Duff, teams up with world-class shooter and all-American girl, Jessie Harrison, for a series unlike any other on the air waves.
Traveling coast to coast, Matt and Jessie unveil one of NRA Foundation's best kept secrets—the multi-million dollar, grassroots fundraising movement making up the heart of the shooting world—Friends of NRA.
friends banquets | nra foundation
Good Luck to Matt and Jessie. Hope your efforts pay off for you...
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Your link to Wisconsin's land, air and water resources
Look who's talking: Milwaukee and Waukesha
Milwaukee and Waukesha officials are edging closer to starting negotiations over Waukesha's request to buy Lake Michigan water from Milwaukee.
On Friday, Waukesha Water Utility General Manager Dan Duchniak provided Milwaukee Mayor Tom Barrett with more details on where Waukesha could expand its water service area and the potential for development beyond its current boundaries in the next four decades.
A map and several reports summarizing population and other demographic information from Waukesha's municipal neighbors that might request water from that city in the future were prepared in the wake of an April 17 meeting between Duchniak and Patrick Curley, Barrett's chief of staff.
The map shows an enlarged Waukesha water supply area as recommended by the Southeastern Wisconsin Regional Planning Commission. Here is a link to the map: http://media.jsonline.com/documents/servicearea.pdf.
Next week, Duchniak expects to meet with staff of the Milwaukee Water Works to exchange information on how to connect Waukesha to Milwaukee's water distribution system, he said in an email distributed Friday to Waukesha Water Commission members and Mayor Jeff Scrima.
The meetings are the start of a dialogue between Milwaukee and Waukesha and are not the beginning of negotiations over a water deal, Barrett spokeswoman Jodie Tabak said. Curley did not respond Friday to a request for an interview.
In Friday's correspondence with Barrett, Duchniak restates his viewpoint that a decision by Milwaukee to sell Lake Michigan water to Waukesha would not spur unbridled growth and give Waukesha a competitive advantage over Milwaukee in attracting new employers.
Only 15% of Waukesha's expanded water service area is available for new development, he says in the letter to Barrett. Here is a link to the letter: http://media.jsonline.com/documents/barrettletter.pdf.
"For the most part, any new water service area beyond Waukesha's current service area will serve areas that are already developed, but are using private wells for water supply," he says.
Duchniak also repeated the assurance that any lakeshore community selling water to Waukesha would gain an economic advantage from the additional revenue at the same time that Waukesha's water rates are expected to more than double due to the costs of obtaining a new supply.
Any formal negotiations over a water deal must wait for the completion of three long-delayed reports requested by the Milwaukee Common Council in July 2011. Reports from the Milwaukee Water Works, Department of City Development and Legislative Reference Bureau are pending, city officials said Friday.
Waukesha is seeking the permission of Wisconsin and each of the other seven Great Lakes states to buy several million gallons a day of lake water so that it can abandon a ground water supply tainted with unsafe levels of radium.
Waukesha identifies Milwaukee, Oak Creek and Racine as three potential suppliers in a request for lake water under review by the state Department of Natural Resources. The DNR is drafting an environmental impact analysis of the proposal and will hold public hearings on its findings this spring or summer.
If Wisconsin approves the request, DNR officials said they will require Waukesha to have negotiated a purchase agreement with one of the three possible suppliers before the request is forwarded to the other Great Lakes states.
Waukesha has been negotiating with Oak Creek and Racine for several months.
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China Environment Publications
CES 8 Special Report, pp.202-211:Jul 07, 2011
River Basin Commissions as a Mechanism for Mitigating and Resolving Conflicts By Irene B. Brooks and Liu Hongxia Special Report Box: Transboundary River Tensions--Opportunities for Collaboration By Juli S. Kim and Michael Murphy more
CES 9 Forward and Table of ContentsJul 07, 2011
CES 9 Special Report, pp. 135-163Jul 07, 2011
The China Environmental Health Project By Jennifer Turner & Linden Ellis Spotlight on NGO Activism: The Jane Goodall Institute Roots & Shoots Program in China By April Nigh Water Resource Challenges in the Karst Regions of Southwest China By Chris Groves Spotlight on NGO Activism: Natural Resources Defense Council China Program By Alex Wang Reaching Out to the Community in Rural Yunnan's Karst Region By Amelia Chung Spotlight on NGO Activism: Catalyzing Participatory Governance From the Grassroots to the County Seat By Amelia Chung Clearing the Air: Promoting Clean Coal Technology and Environmental Health Studies in Huainan City By Wei-Ping Pan Feature Box: Coal City: Measures to Control Urumqi's Health-Threatening Air Pollution By Zhang Chen & Rui Li-hong more
CES 7 Chinese NGOs, GONGOs and Student OrganizationsJul 07, 2011
CES 5 Feature Article, pp. 45-58; Commentaries/Notes From the Field, pp. 59-92Jul 07, 2011
Feature Article, p. 45-58: New Partners or Old Brothers? GONGOs in Transnational Environmental Advocacy in China by Fengshi Wu Commentaries/Notes From the Field, p. 59-92: Looking into a Microcosm of China's Water Problems: Dilemmas of Shanxi--A High and Dry Province by Jih-Un Kim, p.59 Networking for Development of Legal Assistance to Pollution Victims in China by Kenji Otsuka, p.63 Xinjiang: A Trip to the New Territory by Wen Bo, p.66 China's Nature Reserves: Protecting Species or Generating Profits? by Lawrence Glacy, p. 69 The Yunnan Great Rivers Project by Ou Xiaokun, p.74 Environmental Action as Mass Campaign by Jane Sayers, p.77 Spray-Painting Change? Beijing's Green Olympics, NGOs and Lessons Learned from Sydney by Timothy Hildebrandt, p.80 Brick by Brick: Improving the Energy and Environmental Performance of China's Buildings by Robert Watson and Barbara Finamore, p.86 Lean and Green: Boosting Chinese Energy Efficiency through ESCOs by Pam Baldinger, p.90 Energy and Environmental Performance of China's Buildings by Robert Watson and Barbara Finamore, p.86Lean and Green: Boosting Chinese Energy Efficiency through ESCOs by Pam Baldinger, p.90 more
CES 10 Feature Article, pp. 5-32Jul 07, 2011
Feature Article, pp. 5-32 Seeing Through the Smog: Understanding the Limits of Chinese Air Pollution Reporting By Steven Q. Andrews Feature Box: Water and Environmental Health: BSR Takes Action to Break Southern China's Water Crisis By Linda Hwang more
English Sections 2-4Jul 07, 2011
CES 8 Feature Article, pp.3-26:Jul 07, 2011
Legal Advocacy in Environmental Public Participation in China: Raising the Stakes and Strengthening Stakeholders By Allison Moore and Adria Warren Feature Box: Yuanmingyuan's Shifting Landscape: From Emperor's Resort to a Public Green Space By Linden Ellis Feature Box: The Asia Program at the Woodrow Wilson Center more
China Environment Series 11(2010/2011)Nov 10, 2010
China's success in promoting clean energy technology has been a hot story over the past year as the China Environment Forum team pulled together this special Energy and Climate issue of the China Environment Series. We ambitiously aimed to create; and hope our readers think we succeeded;in creating an issue that takes a snapshot of major energy trends in China and understand some of the complexities in the U.S.-China energy and climate relations. This eleventh issue is our biggest yet, due not just to our inability to say no to so many great paper proposals, but also because of the dynamism in clean energy developments in China and many exciting developments in U.S.-China energy cooperation;in both government, NGO, and business spheres. We hope you enjoy this issue! This special Energy and Climate issue was made possible through a grant from the Rockefeller Brothers Fund, as well as support from the Blue Moon Fund, USAID, Vermont Law School, Western Kentucky University, and the ENVIRON Foundation. more


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Senior basketball player Levi Randolph and senior tennis player Emily Zabor were named the winners of the Paul W. Bryant Award given annually to the top male and female student-athlete
Three Crimson Tide golfers finish among top-10 individuals
Lovelady finishes in seventh overall to lead the Crimson Tide
Providing Vital Guidance on Career Development
Three Crimson Tide golfers finish among top-15 individuals
Men's Golf Wins the Jerry Pate Intercollegiate Championship
National Championship Recap
Men's Golf National Championship Highlights
OUTLOOK The lone junior on the team who will be looked on as this year's leader after the departure of three starters from a year ago ... an elite national recruit out of the state of Alabama who gives the Crimson Tide six in-state players on the team's nine-man roster ... earned a spot in the Alabama lineup in 2013-14, making 10 starts in 12 events ... played in three events in the fall of 2011 before an injury ended his season ... was granted a medical hardship that gave him another season of eligibility ... made four starts and played in six events as a redshirt freshman in 2012-13 ... has a 73.05 scoring average over 56 career rounds ... has the raw talent and all-around skills to be very successful at Alabama and beyond.2014-15 (JUNIOR) Started all four tournaments during the fall season for the Crimson Tide ... was second on the team with a scoring average of 71.50 ... logged a team-high seven rounds of par or lower and placed in the top-10 twice ... ranked as the 56th-best individual in collegiate golf by the Golfweek/Sagarin Performance Index ... had 10 of 12 rounds count toward the team score ... put together the lowest round by an Alabama golfer in the fall with a 64 during the final round of the Jerry Pate National Intercollegiate ... was the Crimson Tide's top performer and set his own 54-hole personal best at the Shoal Creek Intercollegiate with a second place finish by shooting 70-66-70 (10-under 206) ... opened the season with a 3-over 219 at the Carpet Capital Collegiate while finishing tied for 41st ... shot 13-over 223 and finished tied for 25th at the Olympia Fields/Illini Invitational ... rounded out the fall season tying for eighth at even-par 210 at the Jerry Pate National Intercollegiate and put together his finest collegiate round by shooting 64 in the final round. 2013-14(SOPHOMORE) Made 10 starts in 12 events for the Crimson Tide, helping Alabama to its second straight NCAA Championship ... fifth on the team in scoring average at 72.27 (2,168 strokes/30 rounds) ... finished wiht three top-10 finishes with a tie for third at the NCAA Auburn Regional ... had 13 rounds of par or better this season and five in the 60s ... counted in 22 of 30 rounds this season (.733) ... ranked 68th in the final Golfweek/Sagarin Performance Index and 66th in the last Golfstat.com Head-to-Head Player Rankings ... recorded the first hole-in-one of his collegiate career with an ace on the 11th hole at the Vanderbilt Legends Club in the second round of the Mason Rudolph Championship ... opened the season at the JPNI where he shot 1-over 211 with an first-round 68 for a seventh-place finish ... tied for 28th at the Isleworth Collegiate, shooting 79-68-73 for a 4-over 220 ... closed the fall with a tie for 25th at the Gifford Collegiate firing a 6-over 216 (73-70-73) ... began the spring with a 3-under 213 at the Puerto Rico Classic with rounds of 70-76-67 to tie for 22nd ... tied for 34th at the Southern Highlands Collegiate Masters (78-75-75/228) and 49th at the Linger Longer Invitational (71-76-79/226) ... registered his second top 10 of the season by finishing eighth at the Mason Rudolph Championship ... fired rounds of 75-70-68 for and even-par 213 ... tied for 43rd at the SEC Championship at 7-over 217 (73-71-73) ... turned in one of his best performances of the season with a 3-under 213 at the NCAA Auburn Regional Championship ... his tie for third was a career-best finish ... carded rounds of 72-72-70 ... tied for 35th at 1-over 211 in the stroke play portion of the NCAA Championships with rounds of 67-73-71 ... counted in all three rounds for the Tide who tied for second in the stroke play ... was 1-2 in match play, earning a win over LSU's Smylie Kaufman, 1 up, in the semifinals ... fell 2 up to SMU's Harry Higs in the quarters and battle Big 12 Player of the Year Wyndam Clark of Oklahoma State in the finals, making an impressive comeback before eventaully falling 3 and 1. 2012-13 (REDSHIRT FRESHMAN) Played is six events and started four times for the Crimson Tide ... posted a 74.12 scoring average over 17 competitive rounds ... fired three rounds of 69 and four rounds at par or better ... tied for 16th at the Puerto Rico Classic, carding rounds of 69, 76, 69 for a career-low 2-under 214 ... top finish was a tie for ninth at the Savannah Quarters Invitational, after shooting 7-over 223 ... started Alabama's first event of the 2012-13 season at the Carpet Capital Collegiate, finishing 26th at 5-over 221 (73-72-76) ... posted a final-round 69 at the Isleworth Collegiate Invitational ... shot 6-over 222 in starting role at the Schenkel Invitational, with rounds of 75, 74 and 73 ... competed as an individual at the Linger Longer Invitational, finishing 46th at 7-over 151 ... finished second at the Dixie Amateur, shooting a personal best 63 in the third round ... finished third at the 2013 Alabama State Amateur ... tied for fourth at The Players Amateur in Bluffton, S.C. 2011-12 (FRESHMAN) Started the first three tournaments before an injury derailed his freshman season ... posted two top-25 finishes and averaged 73.67 shots per round, which ranked fifth on the team ... counted toward the team score in 8-of-9 rounds... carded a 2-over-par 218 in his collegiate debut at the Carpet Capital Collegiate, finishing tied for 21st ... placed 25th at the Olympia Fields Fighting Illini Invitational, shooting 10-over 220 ... carded a 225 (+15) at the Jerry Pate National Intercollegiate to finish 39th ... named to the SEC First-Year Academic Honor Roll ... granted a medical hardship for the 2011-12 season. PREP HIGHLIGHTS Two-time Rolex Junior All-American, garnering honors in 2009 and 2010 ... listed No. 13 nationally in the Golfweek Junior Boys Rankings ... 2008 and 2011 Birmingham News Metro Player of the Year ... Alabama High School Athletics Association Class 6A medalist in 2008 and 2011 ... led his team to the 2011 Alabama 6A State Championship title ... 2011 Birmingham Kiwanis Club Golfer of the Year ... led his Mountain Brook High School team to a runner-up showing in 2008 state tournament ... low competitive round as a junior golfer was a 64 at the PGA Junior at Shoal Creek in Birmingham ... also shot 64 at the 2011 Dogwood Invitational to tie his low competitive round as a junior ... placed third at the 2012 Palmetto Amateur ... finished second to classmate Justin Thomas at the 2009 Footjoy Invitational, where he carded rounds of 69, 67, 69 and 69 ... shot 69 and 65 at the 2010 USGA Junior Amateur Qualifier ... won the 2010 Willow Point Invitational and finished fifth at the 2011 Dixie Amateur ... advanced to the round of 16 at the 2008 USGA Junior Amateur ... set the course record at Vestavia Hill Country Club with a 61 in August of 2012 ... medalist at the 2008 West Alabama Classic. PERSONAL INFORMATION Son of Tim and Kitty Lovelady ... has one sister, Annie ... was involved with FBLA, Spanish Club, Young Republicans and Future Business Leaders of America at Mountain Brook ... a pre-business major at Alabama.


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Communicating practical messages from the Bible to the heart and home
Lisa J. Davis has formally served in church ministry work since 2009 but has been involved with speaking and Bible teaching for much longer. She held her first women's conference, Diamonds in the Rough, in December of 2008, encouraging women to move "From Soot to Shine." Prior to this, Lisa taught adult Sunday school as well as worked alongside others in teaching youth church services. Lisa has been a frequent teacher for midweek Bible study and has been a requested speaker for prayer breakfast worship and women's retreats. She currently works in education, in which she has served families in both public and private school settings for over eleven years. She resides in Texas with her husband, Michael, and their two daughters.


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Gallery Folders
Shingeki no kyojin
Avatar LOK
Avatar LAB
Anatomy and drawing tips
Grim Reaper
nothing to say to your Gallery (it's awesome btw) but i just read through everything for the lols xD
Bruh, this gallery is like drugs.
Your now my role model
your gallery^^
Hey there! ^___^
Your art is really amazing, you have a great talent <3
But I wanted to tell you that there's a problem, someone steal some of your art: maskedman08.deviantart.com/gal…
I love your merpeople they are interesting , inspiring, i love them because of how different and original they are i never seen any merperson so unique
Ffff, I love your art so much!!
so super , beautifil, soulful amazing pieces of art you did and this is why the line work is beautiful the line weight strong, clean and very smooth .the all around color it a masterpiece amazing digital color you pull it off greatly with super beautiful bright colors .. great use of warm and cool colors e 50/ 50 with it make the image very hd i love it. great mix with the all the color and it as a mood to it i love the whole thing to the rich color layout with the sharp edge to smooth edge it make it pop out to the screen it willing catch my eye and the flow of it again amazing beautiful pieces of art
Your drawings are so perfect!! I was wondering if you can make some Bleach fanart too? I'd be so happy ;__; Thank yoiu!
I really adore that in your populars are some personal work ( like ying yang fishes ) cause uh - tutorial,tutorials, fanarts..and OH i dont see SnK in first page of populars :0
Wow, I never thought I would find someone who draws everything I like--and extremely well, on top of that. Sorry for harassing you with all the faves, haha. Amazing gallery.