Patent Application: US-34004408-A

Abstract:
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 .

Description:
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 . colored glass rods : mint green for the visible mode calibration , and translucent blue for the ultraviolet ( uv ) mode calibration . solid calibration adapter : black diacetyl plastic machined to fit the instrument . glass rods are glued into the solid calibration adapter and sealed with a plastic cap , ( see fig1 ). this recipe makes a 10 × stock solution . it must be diluted 1 : 10 into distilled water to make a 1 × working solution before being used . 30 ml 10 × sodium acetate buffer : ( 13 . 6 grams sodium acetate in 100 ml ddh 2 o ) 1 ) remove the mini cell receptacle from the instrument . 2 ) be sure that the instrument is in “ a ” mode ; the letters uv should appear in the lower left corner of the screen . 3 ) if the instrument is not in “ a ” mode , press the a / b key on the instrument keypad . 4 ) press the cal key on the keypad ; when prompted , press enter to continue . 5 ) when asked to insert the blank , insert the solid standard labeled “ a ” into the instrument with the letter “ a ” facing down and to the right . 6 ) when asked to insert the “ cal ”, insert the solid standard labeled “ a ” into the instrument so that the letter a is facing towards you , and the white cap is on top . 1 ) follow all the instructions for calibration in “ a ” mode , making sure that that the instrument is now in “ b ” mode , and that the solid standard labeled “ b ” is now being used . 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 fig2 . 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 fig3 . 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 r 2 values of 0 . 985 , 0 . 987 , and 0 . 962 respectively , p & lt ; 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 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 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 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 . 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 . 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 . fig6 shows the correlation between the easy count values and the cells / ml results of the hemacytometer . a statistically linear relationship was found between cell counts obtained by the asbc standard method of microscopic examination using a hemacytometer and values obtained using the easy count , r 2 = 0 . 985 . fig7 illustrates the linear correlation found between the easy count method and the asbc method for methylene blue staining . a statistically linear relationship was found between the easy count , and hemacytometer counts corrected for viability , r 2 = 0 . 987 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 . a linear relationship was found between the easy count and slide culture for yeast viability , as shown in fig8 . 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 . cell growth was measured during laboratory and brewery scale fermentations using both the asbc method for hemacytometer counts , and the easy count method . fig9 shows cell growth tracked by both methods during laboratory scale fermentation . fig1 is an example of a brewery scale fermentation tracked by both methods . 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 fig1 . 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 fig7 .). a percent error of 3 % between operators gives a range between 191 - 203 million cells / ml , a difference of only 12 million cells / ml . the very low error associated with the performance of the easy count provides much more reliable information to the brewer . 1 . mochaba , f . et al , practical procedures to measure yeast viability and vitality prior to pitching . j . am . soc . brew . chem . 56 ( 1 ): 1 - 6 , 1998 . 2 . o &# 39 ; connor - cox , e . et al , methylene blue staining : use at your own risk . tech . q . master . brew . assoc . 34 : 306 - 312 , 1997 3 . carvell j . p . et al developments in using off - line radio frequency impedance methods for measuring the viable cell concentration in the brewery . j . am . soc . 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